Chronic pouchitis after ileal pouch-anal anastomosis: responses to butyrate and glutamine suppositories in a pilot study.
Nonspecific, idiopathic inflammation of ileal pouch mucosa ("pouchitis") after ileal pouch-anal anastomosis is a common complication of this surgical approach. The epithelium of the pouch is ileal, but variable degrees of colonic metaplasia are natural sequelae of construction of such a pouch. One hypothesis is that pouchitis is caused by a deficiency of epithelial nutrition. Thus, a lack of butyric acid (the principal metabolic fuel of colonocytes) or of glutamine (the main fuel of enterocytes) may develop. In this study, our aims were to determine the concentration of total short-chain fatty acids in random stool samples obtained from patients with an ileal pouch-anal anastomosis with and without pouchitis and to test the therapeutic effects of butyrate and glutamine suppositories on pouchitis. During the study, all conventional therapy for pouchitis was discontinued. For 21 days, 9 patients participated in a butyrate trial, and 10 patients were treated with glutamine. Total concentrations of fecal short-chain fatty acids were significantly less in patients with pouchitis than in those without pouchitis. During treatment, 6 of the 10 patients who received glutamine had no recurrence of symptoms, but only 3 of the 9 patients who received butyrate responded similarly. Hence, further studies of glutamine in the treatment of pouchitis seem warranted.


[Magnetic resonance in the identification and assessment of extent and severity of bone contusion damage: comparison of several types of imaging (SE/T1, GE/T2 and SE/T1) with fat suppression].
Bone marrow contusion is a common cause of chronic joint pain in the post-traumatic knee; it is usually associated with meniscal and/or ligament damage but it may also be the only abnormal sign. In this condition MRI plays a fundamental role because it is the only imaging technique which can diagnose it and accurately assess its extent and any cortical bone and/or cartilage involvement. We investigated the diagnostic potentials of 3 MR sequences in identifying and assessing the extent of bone contusion. January, 1994, to December, 1997, we performed 539 MR examinations of the knee on 478 patients with chronic joint pain following trauma. We used T1-weighted SE, T2*-weighted GE and T1-weighted SE sequences with fat saturation (fs), paying great attention to technical parameter setting. The images were retrospectively reviewed by groups of radiologists using 2 semi-objective evaluation systems: a qualitative assessment to grade the ease of detection of the contusion with each sequence and a quantitative assessment of contusion extent and of osteochondral involvement, if any, again for each sequence. The qualitative analysis showed that GE and SE-fs sequences were superior to the conventional ones in identifying bone contusion foci; GE sequences tended to overestimate the number of poorly depictable lesions relative to SE-fs ones. The quantitative analysis showed that the conventional sequences tend to underestimate the foci extent, while GE and SE-fs sequences performed nearly the same in the assessment of high-grade lesions. The number of low-midgrade lesions was overestimated by GE sequences relative to SE-fs ones. GE and SE-fs sequences permit accurate studies of bone contusions. Diagnostic efficacy is increased by enhancing the signal from non-fatty structures within a tissue rich in fat: the quantitative reduction of the non-fatty structural component and/or its replacement with abnormal components is thus best shown. Fat-suppression sequences were more accurate in defining lesion size, which is also related to low susceptibility to the artifacts from extrinsic and especially intrinsic structural inhomogeneities. In contrast, GE sequences demonstrated high susceptibility, which caused poorer definition of the contusion borders and, consequently, overestimation of the lesion size.


Retinoic acid 4-hydroxylase-mediated catabolism of all-trans retinoic acid and the cell proliferation in head and neck squamous cell carcinoma.
All-trans retinoic acid (RA) can be catabolized to polar metabolites by microsomal P450s (P450). The aim of this study was to confirm if retinoic acid 4-hydroxylase (CYP26) is a P450 induced by RA and to investigate the role of cellular RA binding proteins (CRABPs), using a slow catabolizer, AMC-HN-4, and a rapid catabolizer, AMC-HN-6. Also, we analyzed the effect of RA catabolism on cell proliferation of head and neck squamous cell carcinoma (HNSCC) in vitro and in vivo. Both cell lines weakly expressed CYP26 and CRABPs, but RA induced CYP26 only in AMC-HN-6. The sensitivity to RA was variable by the amount of CYP26, and the rapid catabolism by CYP26 made AMC-HN-6 resistant to RA in vitro. In addition, The RA had a stronger effect on the inhibition of tumor growth of AMC-HN-4 than that of AMC-HN-6 in vivo. Conclusively, the CYP26 activity might be one essential factor for the RA sensitivity, but in cells showing induction of CYP26, the RA sensitivity is inversely related to the rate of RA catabolism.


Analysis of Glutathione in Biological Samples by HPLC Involving Pre-Column Derivatization with o-Phthalaldehyde.
Glutathione (GSH) forms conjugates with polyamines in prokaryotes and eukaryotes. There is also evidence suggesting cross-talk between GSH and polyamines to regulate cellular homeostasis and function, particularly under the conditions of oxidative stress. Because of its versatile roles in cell metabolism and function, a number of high performance liquid chromatography (HPLC) methods have been developed for glutathione analysis. Here, we describe our rapid and sensitive method for the analysis of GSH and the oxidized form of glutathione (GS-SG) in animal tissues and cells by HPLC involving pre-column derivatization with o-phthalaldehyde (OPA). OPA reacts very rapidly (within 1 min) with S-carboxymethyl-glutathione at room temperatures (e.g., 20-25 °C) in an autosampler, and their derivatives are immediately injected into the HPLC column without any need for extraction. This method requires two simple steps (a total of 15 min) before samples are loaded into the autosampler: (a) the conversion of GS-SG into GSH by 2-mercaptoethanol; and (b) the oxidation of GSH by iodoacetic acid to yield S-carboxymethyl-glutathione. The autosampler is programmed to mix S-carboxymethyl-glutathione with OPA for 1 min to generate a highly fluorescent derivative for HPLC separation and detection (excitation wavelength 340 nm and emission wavelength 450 nm). The detection limit for GSH and GS-SG is 15 pmol/ml or 375 fmol/injection. The total time for chromatographic separation (including column regeneration) is 16 min for each sample. Our routine HPLC technique is applicable for analyses of cysteine and cystine, as well as polyamines and GSH-polyamine conjugates in biological samples.


[The use of a fatty emulsion for determining blood lipids in parenteral nutrition].
The author has investigated the possibility of using as the reference agent in estimation of the resorption rate from vascular bed of fatty emulsions the very agent that is introduced or the oil, one of the ingredients of this agent. Fatty emulsions for parenteral nutrition intralipid (Sweden), lipofundin-C (Finland), venolipid (Japan), lipidin and lipidin-2 (USSR), and specially purified sunflower oil were used to plot the calibration curve.


Case report: Periorbital intraosseous hemangiomas.
Hemangiomas are hamartomatous proliferation of vessels. Intraosseous hemangiomas of the facial bones are rare and most commonly involve the zygoma, maxilla, mandible, and the nasal bones. A "sunburst" pattern is a typical appearance on CT scan and MRI and therefore a biopsy is not always necessary. Surgery is usually performed in symptomatic cases. The authors describe five typical periorbital intraosseous hemangiomas with a brief review of literature.


Photoreceptor cells with unusual functional properties on the ventral nerve of Limulus.
Normal photoreceptor cells on the ventral nerve of Limulus respond to a moderately intense flash with a large receptor potential or current. Occasionally, cells are found in which the same flash evokes only a small receptor potential or current. Our investigations reveal physiological reasons for the poor light sensitivity in these "unusual cells." In unusual cells prolonged illumination with intense light evokes a step-like inward current with an amplitude of some nanoamperes, but without a large transient peak. The current appears to be summed up of single photon responses with amplitudes smaller than about 50 pA. Their time course is similar to that of small single photon responses forming the so-called macroscopic C1 component in normal cells. The macroscopic current evoked by an intense flash has slow activation and deactivation kinetics and reaches a saturated amplitude of about 4-5 nanoamperes. The light-intensity dependence of the current evoked by flashes or by prolonged illumination has a slope of about 1 in log-log plots. The decay kinetics of the current is similar to that of the C1 component measured in normal cells after the block of the C2 component. Occasionally, the step-like current is superposed by large standard bumps. These bumps are blocked by the Ca2+-ATPase inhibitor cyclopiazonic acid, while the sustained inward current persists. We conclude that in unusual cells the light-activated current is identical to the C1 component of normal cells. The phospholipase C pathway that in normal cells presumably gives rise to the C2 component functions only with a low efficiency in unusual cells.


Differential sensitivities of triple-negative breast cancer stem cell towards various doses of vitamin C: An insight into the internal antioxidant systems.
Cancer stem cells (CSCs) are quiescent and self-renewing, having low levels of reactive oxygen species (ROS), and are responsible for cancer recurrence after chemotherapy and radiotherapy. However, the interplay between the ROS production and scavenging from the oxidative stress has never been studied in breast CSCs. In this present study, we have investigated the cellular energetics of two triple-negative breast cancer stem cells (MDA-MB-231 and MDA-MB-468) treated with two pharmacological doses of vitamin C (10 and 20 mM) that generated ROS. Our results indicate a differential behavior of ROS scavenging by both the CSCs. MDA-MB-468 CSCs exhibited higher resistance to ROS induced damage owing to the higher antioxidant activity, lower mitochondrial damage, and less decrease in membrane potential (ΔΨm ) as compared with MDA-MB-231 CSCs. Moreover, MDA-MB-231 CSCs exhibited an intrinsic apoptosis pathway by activating the cytochrome c, caspase-9, 3, 7, and cleaved PARP upon treatment with vitamin C. This data suggests a possible strategy for targeting breast CSCs using vitamin C. Taken together, the CSCs from MDA-MB-231 could be easily targeted by high/pharmacological doses of vitamin C (≥20 mM) thereby indicating a less robust internal antioxidant machinery.


Chin bone harvesting for autogenous grafting in the maxillary sinus: a clinical report.
The sinus lift has become a common dental procedure when insufficient bone exists to support endosseous dental implants. The goal of the procedure is to augment existing bone in the posterior maxilla. Various resorbable and nonresorbable allograft, xenograft, and synthetic materials and mixtures, with and without autogenous bone, have been used for sinus augmentation. The authors prefer a mixture of autogenous bone with resorbable bovine xenograft, utilizing as much autogenous bone as possible. The chin offers an ideal site for bone harvesting because of its advantages over other potential sites, such as iliac crest, rib, and calvarium. These advantages include ease of accessibility, relatively simple office-based surgery, minimal morbidity, and the benefits of utilizing intramembranous versus endochondral bone. This article describes the harvesting and the sinus lift procedures. The learning objective of this article is to present this particular procedure using a clinical report to illustrate.


The meaning of self-injury and overdosing amongst women fulfilling the diagnostic criteria for 'borderline personality disorder'.
To explore the meanings of self-injury and overdosing, and the relationship of each to the other, for women who have fulfilled the diagnostic criteria for borderline personality disorder. Four women with a history of both self-injury and overdosing were recruited through a local psychotherapy service and interviewed following a semi-structured framework. The interview transcripts were systematically analysed utilizing interpretative phenomenological analysis. The analysis lead to the construction of three interrelated themes: (a) The context of distress. Examples were inclusive of temporally distal factors such as childhood abuse and loss, and proximal factors ranging from the interpersonal (e.g., conflict) through to the intra-personal (e.g., negative thoughts relating to self). (b) The progressive management of distress. An overdose was depicted as a last resort once self-injury had become insufficient a means of self-help to manage feelings of desperation and isolation. (c) Ambivalence in relation to death. The clearly stated intention of an overdose was 'to die'. However, both words and (descriptions of) deeds suggested considerable ambivalence in relation to this--overdoses appearing to depict an attempt to resolve unbearable feelings through an unarticulated 'cry for help' rather than suicide completion. The overall impression gained was of a group of women in a world of problematic relations (to both self and others) struggling to know how best to attract support and understanding. In discussion, links are made to the literature on reflectivity/reflexivity through Fonagy's notion of 'mentalization'.


Visual impairment and objectively measured physical activity and sedentary behaviour in US adolescents and adults: a cross-sectional study.
To compare levels of physical activity and sedentary time in a representative sample of US adolescents and adults with and without visual impairment. Cross-sectional analyses were carried out using data from the National Health and Nutrition Examination Survey. The study population consisted of 6001 participants (adolescents n=1766, adults n=4235). The present analysis aggregated data from 2003 to 2004 and 2005-2006. Objective physical activity and sedentary behaviour assessment was conducted over 7 days. Distance visual acuity was measured for each eye in all participants 12 years and older. Participants' vision was categorised as: normal vision, uncorrected refractive error, non-refractive visual impairment. We estimated the sex-specific linear associations between presenting vision and objectively measured physical activity and sedentary patterns using adjusted generalised linear models in adolescents and adults. Adolescents with uncorrected refractive error and non-refractive visual impairment did not accumulate higher levels of sedentary time or lower levels of moderate-to-vigorous physical activity (MVPA) compared with those with normal vision. We observed no association between vision status and accelerometer measured MVPA in adults aged 20-49 years. We observed more time spent sedentary among females 20-49 years old with non-refractive visual impairment compared with those presenting normal vision (mean difference 329.8 min/week, 95% CI: 12.5 to 647.0). Adults 50 years and older with non-refractive visual impairment appeared to accumulate less lifestyle physical activity, particularly in women (mean difference -82.8 min/week, 95% CI: -147.8 to -17.8). Adult women with non-refractive visual impairment have lower levels of lifestyle physical activity and higher levels of sedentary time than those with normal vision. Taken together, these findings highlight the need for interventions to promote physical activity and reduce sedentary time in adult populations with visual impairment, specifically adult women.


Responses to microinjections of endomorphin and nociceptin into the medullary cardiovascular areas.
1. Cardiovascular effects of microinjections of nociceptin and endomorphin-2 into the following medullary areas were studied in urethane-anaesthetized rats: chemoreceptor projection site (CPS), intermediate portion of the nucleus tractus solitarius (I-NTS), caudal ventrolateral medullary depressor area (CVLM) and rostral ventrolateral medullary pressor area (RVLM). 2. Microinjections of nociceptin or endomorphin-2 (0.6 mmol/L each) into the CPS and RVLM elicited depressor and bradycardic responses, whereas similar injections into the I-NTS and CVLM elicited pressor and tachycardic responses. 3. The mechanism of cardiovascular responses to microinjections of these opioid peptides into different medullary areas involved in cardiovascular function can be postulated as follows: the direct effect of nociceptin and endomorphin-2 on neurons is usually inhibitory. Because the activation of CPS and RVLM by microinjections of L-glutamate results in pressor and tachycardic responses, inhibition of these areas by nociceptin and endomorphin-2 elicits depressor and bradycardic responses. Similarly, activation of neurons in the I-NTS and CVLM by microinjections of L-glutamate elicits depressor and bradycardic responses. Therefore, inhibition of these areas by microinjections of these opioid peptides elicits an increase in blood pressure and heart rate.


Biomechanical Assessment of a Novel Sharp-Tipped Screw for 1-Step Minimally Invasive Pedicle Screw Placement Under Navigation.
The objective of this study was to assess the pullout force of a novel sharp-tipped screw developed for single-step, minimally invasive pedicle screw placement guided by neuronavigation compared with the pullout force for traditional screws. A total of 60 human cadaveric lumbar pedicles were studied. Three different screw insertion techniques were compared: (A) Jamshidi needle and Kirschner wire without tapping; (B) Jamshidi needle and Kirschner wire with tapping; and (C) sharp-tipped screw insertion. Pullout tests were performed at a displacement rate of 10 mm/min recorded at 20 Hz. Mean values of these parameters were compared using paired t tests (left vs right in the same specimen): A vs B, A vs C, and B vs C. Additionally, 3 L1-L5 spine models were used for timing each screw insertion technique for a total of 10 screw insertions for each technique. Insertion times were compared using 1-way analysis of variance. The mean pullout force for insertion technique A was 1462.3 (597.5) N; for technique B, it was 1693.5 (805.0) N; and for technique C, it was 1319.0 (735.7) N. There was no statistically significant difference in pullout force between techniques (P > 0.08). The average insertion time for condition C was significantly less than that for conditions A and B (P < 0.001). The pullout force of the novel sharp-tipped screw placement technique is equivalent to that of traditional techniques. The sharp-tipped screw placement technique appears biomechanically viable and has the advantage of saving time during insertion. Single-step screw placement using high resolution 3-dimensional navigation has the potential to streamline workflow and reduce operative time.


Procalcitonin, a valuable biomarker assisting clinical decision-making in the management of community-acquired pneumonia.
Community-acquired pneumonia (CAP) is a leading cause of mortality, morbidity and hospital admission, which places strain on our healthcare system. Procalcitonin (PCT) is a biomarker of bacterial infection which may help gauge the severity and prognosis of patients with CAP. In addition to clinical predictors, PCT may assist in decisions pertaining to timing of discharge from hospital and the discontinuation of antibiotics. This study aimed to determine the predictive role of PCT measurement in reducing hospital admissions, length of stay (LOS) and antibiotic (AB) usage in patients with CAP. A prospective, single-blinded, externally controlled study of consenting adult patients admitted with CAP. PCT levels were obtained on day 1 and day 3 (when indicated). Investigator-evaluated clinical parameters, together with results of PCT levels, determined the timing of oral AB switch and discharge from hospital. This process was compared against standard practice, but was not actually implemented, for the purpose of this study. Sixty patients were included in the study. The mean age was 66.5 ± 21.2 years (56.3% male). The average Pneumonia Severity Index was 93 ± 39 (class IV) and the median CURB-65 was 2. The mean LOS for the standard practice cohort was 5.3 ± 4.6 days versus calculated LOS using the PCT guidance pathway of 3.7 ± 2.8 days. (P = 0.00006). Our study supports the hypothesis that by incorporation of PCT levels, hospital admission and LOS in patients with CAP can be reduced. A randomised prospective clinical trial is planned in an attempt to help confirm these findings.


Stenosing Tenosynovitis Due to Excessive Texting in an Adolescent Girl: A Case Report.
We present a case report of stenosing tenosynovitis of the flexor pollicis longus tendon in an adolescent girl who required surgical release after failing conservative measures. The patient had no other risk factors, aside from her excessive texting, which we postulate led to her condition. Although there have been a few reports of tendinitis and tenosynovitis secondary to texting, we believe this is the first in the literature to report trigger thumb requiring surgical release in an adolescent.


Vascular Complications of Systemic Sclerosis during Pregnancy.
Systemic sclerosis (SSc) is a chronic autoimmune disorder characterized by progressive fibrosis of the skin and visceral tissues as well as a noninflammatory vasculopathy. Vascular disease in systemic sclerosis is a major cause of morbidity and mortality among nonpregnant patients with SSc and is even a bigger concern in the pregnant SSc patient, as the underlying vasculopathy may prevent the required hemodynamic changes necessary to support a growing pregnancy. Vascular manifestations including scleroderma renal crisis and pulmonary arterial hypertension should be considered relative contraindications against pregnancy due to the high associations of both maternal and fetal morbidity and mortality. In contrast, Raynaud's phenomenon may actually improve somewhat during pregnancy. Women with SSc who are considering a pregnancy or discover they are pregnant require evaluation for the presence and extent of underlying vasculopathy. In the absence of significant visceral vasculopathy, most women with SSc can expect to have reasonable pregnancy outcomes.


Large-scale expression, purification and characterization of small fragments of thrombomodulin: the roles of the sixth domain and of methionine 388.
Fragments of human thrombomodulin (TM) have been expressed in large quantities in the Pichia pastoris yeast expression system and purified to homogeneity. Fermentation of P. pastoris resulted in yields of 170 mg/l TM. Purification to homogeneity resulted in an overall 10% yield, so that quantities of approximately 20 mg purified fragments can be readily obtained. Smaller fragments of TM, such as the individual fourth or fifth domains, were not active, nor were equimolar mixtures of the two domains. These results demonstrate that the fourth and fifth epidermal growth factor (EGF)-like domains together comprise the smallest active fragment of TM. The fragment containing the fourth and fifth EGF-like domains [TMEGF(4-5)] had 10% the specific activity of rabbit TM. Comparison of the M388L mutant TMEGF(4-5) fragment with the same mutant TMEGF(4-5-6) fragment showed that the fragment with the sixth domain had a 10-fold better Km value for thrombin than the fragment that did not contain the sixth domain; this factor completely accounts for the higher specific activity of the fragments containing the sixth domain. Comparison of the wild-type and M388L mutants showed that the M388L mutation resulted in a 2-fold increase in kcat for the activation of protein C by the thrombin-TM fragment complex, completely accounting for the 2-fold increase in specific activity of these mutant fragments.


Prospective evaluation of the course of disc herniations in patients with proven radiculopathy.
Although surgery is often recommended as the definitive treatment for radiculopathy when definite disc herniation is demonstrated with imaging techniques, complete improvement can occur with nonoperative treatment. However, what happens to the disc in the latter circumstance is not well defined. We report the first prospective study in subjects with proven radiculopathy and definite disc herniation who improve with nonoperative management to determine what occurs to the herniated disc material. Eighteen subjects with lower extremity pain or paresthesia, positive straight leg raising, weakness in a myotomal distribution, reflex asymmetry, or electromyogram evidence of radiculopathy were studied. Subjects were admitted to the study if computed tomography (CT) scanning demonstrated definite disc herniation corresponding to the side and level of the radiculopathy. After complete clinical improvement, repeat CT scan was performed at six to 18 months after the initial study. The CT scans were interpreted separately by two neuroradiologists. Disc herniations were characterized by size (large, moderate, or minimal); the presence of absence of free fragments; and location. Follow-up scans were compared with the original study and characterized as resolved, improved, or unchanged. Fourteen subjects completed the study, an additional three had operative treatment, and one refused repeat scanning. Subjects were followed an average of 30.4 months with no recurrence of radicular symptoms during this follow-up period in 13 patients. One had recurrence of symptoms at 21 months and surgery at 26 months. Six follow-up scans (43%) were interpreted as completely resolved, five (36%) as improved, and three (21%) as unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)


Intracranial calcifications on CT.
Computed tomography (CT) is very sensitive for detection and localization of intracranial calcifications. We reviewed in this pictorial essay the diseases associated with intracranial calcifications and emphasized the utility of CT for the differential diagnosis.


Domain analysis of the Nematostella vectensis SNAIL ortholog reveals unique nucleolar localization that depends on the zinc-finger domains.
SNAIL transcriptional factors are key regulators during development and disease. They arose early during evolution, and in cnidarians such as Nematostella vectensis, NvSNAILA/B are detected in invaginating tissues during gastrulation. The function of SNAIL proteins is well established in bilaterians but their roles in cnidarians remain unknown. The structure of NvSNAILA and B is similar to the human SNAIL1 and 2, including SNAG and zinc-finger domains. Here, we performed a molecular analysis on localization and mobility of NvSNAILA/B using mammalian cells and Nematostella embryos. NvSNAILA/B display nuclear localization and mobility similar to HsSNAIL1/2. Strikingly, NvSNAILA is highly enriched in the nucleoli and shuttles between the nucleoli and the nucleoplasm. Truncation of the N-terminal SNAG domain, reported to contain Nuclear Localization Signals, markedly reduces nucleolar levels, without effecting nuclear localization or mobility. Truncation of the C-terminal zinc-fingers, involved in DNA binding in higher organisms, significantly affects subcellular localization and mobility. Specifically, the zinc-finger domains are required for nucleolar enrichment of NvSNAILA. Differently from SNAIL transcriptional factors described before, NvSNAILA is specifically enriched in the nucleoli co-localizing with nucleolar markers even after nucleolar disruption. Our findings implicate additional roles for SNAG and zinc-finger domains, suggesting a role for NvSNAILA in the nucleolus.


Type and significance of fetal arrhythmias.
Fetal echocardiography was undertaken in 350 high risk pregnant females between 14 to 36 weeks of gestation at our institution. Significant fetal arrhythmias were diagnosed with the help of M-mode and pulsed Doppler echocardiography in 24 pregnant females. Three categories of arrhythmias were observed: (i) atrial or ventricular extrasystoles in 17, (ii) supraventricular tachycardia in 4, and (iii) congenital complete atrioventricular block in 3. The first category of arrhythmia was benign in 100 percent of cases with complete resolution before or just after birth. The second category was completely treatable with antiarrhythmic drugs given to the mother, thus preventing complications of heart failure and hydrops. In the third category, one fetus who had associated severe structural cardiac malformation, died in utero. The other two are doing well postnatally and are on medical follow up. Recognition of arrhythmias and appropriate prenatal treatment will prevent intrauterine death and also help avoid unnecessary operative or premature deliveries.


Long-term results of the total condylar knee arthroplasty. A 15-year survivorship study.
This study reports the 15-year survivorship of 112 consecutive Total Condylar knee arthroplasties that have been followed since 1974. Two endpoints were chosen for survivorship: (1) Revision attributable to septic or aseptic loosening or malalignment. (2) Revision or roentgenographic evidence of component loosening. Life table analysis reveals a 94.1% clinical survivorship at 15 years, with an 90.9% survivorship when roentgenographic failures are included. There were five revisions: one for infection, one for instability, and three for tibial loosening. In addition, two tibiae and one patella were considered roentgenographically loose, but were not symptomatic. As of May 1992, 34 patients with 48 knees are known deceased, 15 knees are lost to follow-up evaluation, and 49 knees are available for clinical evaluation. Follow-up data was available on 62 knees for greater than 11 years. Ninety-two percent had good or excellent results, with 1.6% fair and 6.5% poor. Average range of motion was 99 degrees. The average Hospital for Special Surgery knee score was 85. Roentgenographic study revealed lucencies around 72% of tibiae, but only two components were loose. There was a correlation between body weight and the presence of radiolucencies, and patients who weighed more than 80 kg had the lowest survivorship at 15 years: 89.2% clinical survival and 70.6% clinical plus roentgenographic survival. Total Condylar knee arthroplasty has a 94.6% clinical survival at 15 years, with predictably good clinical results.


Development and evaluation of a teleoncology system for breast cancer during the COVID-19 pandemic.
Background: The present study describes the steps of developing a hybrid teleoncology system to provide treatment plans for breast cancer patients. Materials & methods: This research was conducted in four stages, including developing a proposal for experts, identifying and analyzing system requirements, designing a prototype and implementing and evaluating the final version of the hybrid teleoncology system. Results: The results of the usability evaluation showed that the users evaluated the system at a good level and, in practice, the implemented system was perceived to be useful by specialists in providing treatment plans for cancer patients. Conclusion: The hybrid teleoncology system is a practical alternative to traditional methods for providing treatment plans to breast cancer patients.


[Nephrosclerosis: current status].
Nephrosclerosis is the most typical and widespread renal manifestation of hypertension and can be judged as the pathological hallmark of essential hypertension. Nephrosclerosis is an important and frequent cause of progressive renal disease, however, information in the literature on the risk of developing renal failure in the course of essential hypertension is sparse. Traditionally, nephrosclerosis was thought to result from glomerular ischemia. Alternatively, glomerular sclerosis in hypertension may result from glomerular hyperperfusion or hypertension. Studies in experimental models of renal disease have identified a promising intervention with either Ca antagonists or angiotensin-converting enzyme inhibitors. Application of these therapies to patients with nephrosclerosis should await the results of careful clinical trials.


A case of reversible posterior leukoencephalopathy syndrome caused by transient hypercoagulable state induced by infection.
We report a normotensive case of reversible posterior leukoencephalopathy syndrome caused by transient hypercoagulable state. Hypertension is the main risk factor for reversible posterior leukoencephalopathy syndrome, which is believed to occur as a result of high blood pressure-related dysfunction of cerebrovascular endothelial cells, because it commonly appears in hypertensive emergency. However, in this completely normotensive case, the typical clinical findings of reversible posterior leukoencephalopathy syndrome were triggered by transient hypercoagulable state without any blood pressure variation. The case was successfully treated with anticoagulation therapy using heparin. Thus, this case indicates that reversible posterior leukoencephalopathy syndrome is induced by cerebrovascular endothelial dysfunction, which is induced not only by high blood pressure but also hemostatic dysfunction.


Analysis of recombination rate in female and male gametogenesis in pearl millet (Pennisetum glaucum) using RFLP markers.
Sex as a factor affecting recovered recombination in plant gametes was investigated in pearl millet, Pennisetum glaucum, by using reciprocal three-way crosses [(AxB)xCvCx(A x B)]. The two populations were mapped at 42 loci pre-selected to cover the majority of the genome. No differences in recombination distances were observed at the whole-genome level and only a few individual linkage intervals were found to differ, all in favour of increased recombination through the male. Distorted segregations found in the three-way crosses provide evidence of post-gametic selection for particular gene(s) or chromosome regions. The significance of these results for the design of pearl millet breeding programmes and inheritance experiments, as well as for other experimental strategies, is discussed.


A factor analysis of associations among self-reported immune related symptoms in a large twin sample.
We examined the cumulative prevalences of 22 symptoms thought to reflect immune system function reported in a questionnaire mailed to 7616 Australian twins. The associations between symptoms and demographic variables were expressed in terms of polychoric or polyserial correlations, and a principal components analysis performed. Factors representing underlying propensities respectively to allergic disease, various minor infections, diseases associated with aging such as arthritis, skin disease, and respiratory tract infection were extracted. Possible processes underlying these symptom clusters and the relative strengths and weaknesses of this type of analysis are discussed.


Localized BMP4-noggin interactions generate the dynamic patterning of noggin expression in somites.
Interactions between BMP4 and its inhibitor, noggin, regulate patterning of somites and neural crest. During mesoderm development, noggin mRNA is expressed in the intermediate mesoderm. Upon segmentation, it is detected in the lateral portion of epithelial somites becoming progressively medialized as they mature. In dissociated segments, noggin becomes transiently confined to the dorsomedial lip of the dermomyotome. Here, we investigated the factor(s) that control this lateral-to-medial shift in transcription of somitic noggin. Inhibition of BMP activity in the caudal lateral plate/intermediate mesoderm prevented noggin transcription in the lateral somite. Further rostrally (or later in development), inhibition of tube-derived BMP, but not of Wnt activity, prevented initial noggin expression in the dorsomedial lip of the dermomyotome. Moreover, BMP4 was sufficient to trigger initial expression of noggin even in the absence of ectoderm and/or neural tube, suggesting a direct action on the dorsomedial somite. Thus, the patterns of noggin transcription in somites are directly regulated by BMP4 activities emanating first from the mesoderm and later from the neural tube. Expression patterns of BMP4 and of type IA BMP receptors are spatiotemporally compatible with this lateral-to-medial shift. These results highlight the existence in the neural tube-mesoderm complex of a regulatory loop by which BMP positively regulates transcription of noggin, which in turn represses further ligand activity.


Genome-wide analysis of acute low salinity tolerance in the eastern oyster Crassostrea virginica and potential of genomic selection for trait improvement.
As the global demand for seafood increases, research into the genetic basis of traits that can increase aquaculture production is critical. The eastern oyster (Crassostrea virginica) is an important aquaculture species along the Atlantic and Gulf Coasts of the United States, but increases in heavy rainfall events expose oysters to acute low salinity conditions, which negatively impact production. Low salinity survival is known to be a moderately heritable trait, but the genetic architecture underlying this trait is still poorly understood. In this study, we used ddRAD sequencing to generate genome-wide single-nucleotide polymorphism (SNP) data for four F2 families to investigate the genomic regions associated with survival in extreme low salinity (<3). SNP data were also used to assess the feasibility of genomic selection (GS) for improving this trait. Quantitative trait locus (QTL) mapping and combined linkage disequilibrium analysis revealed significant QTL on eastern oyster chromosomes 1 and 7 underlying both survival and day to death in a 36-day experimental challenge. Significant QTL were located in genes related to DNA/RNA function and repair, ion binding and membrane transport, and general response to stress. GS was investigated using Bayesian linear regression models and prediction accuracies ranged from 0.48 to 0.57. Genomic prediction accuracies were largest using the BayesB prior and prediction accuracies did not substantially decrease when SNPs located within the QTL region on Chr1 were removed, suggesting that this trait is controlled by many genes of small effect. Our results suggest that GS will likely be a viable option for improvement of survival in extreme low salinity.


Mechanical properties of thin polymer films on stiff substrates.
Force-displacement curves have been acquired with a commercial atomic force microscope on thin films of poly(n-butyl methacrylate) on glass substrates in order to examine the so-called "mechanical double layer" topic, i.e. the influence of the substrate on the mechanical properties of the film in dependence of the film thickness. The hyperbolic fit, a novel semi-empirical equation introduced in previous articles, has been further corroborated. The interpretation of this equation has been deepened, yielding a quantitative and demonstrative characterization of the mechanical properties of double layers. Provided that the Young's moduli of bulk polymer and substrate are measured from the deformation curves, this mathematical model permits to fit the deformation-force curves on the double layers and to determine the thickness of the polymer films in wide range (0-200 nm).


Diagnosis of the mucopolysaccharidoses.
The mucopolysaccharidoses (MPSs) often present a diagnostic challenge, particularly for patients who have more slowly progressive disease phenotypes, as early disease manifestations can be subtle or non-specific. However, certain types of bone and joint involvement should always prompt consideration of an MPS diagnosis, such as early joint involvement without classic inflammatory features or erosive bone lesions, claw hand, spinal deformities or dysostosis multiplex. All such patients should be referred to a geneticist or metabolic specialist for diagnostic evaluation. The earlier the diagnosis is made, the better the potential outcome of treatment. Each type of MPS is associated both with deficient activity of a specific lysosomal enzyme that degrades specific glycosaminoglycans (GAGs) and with abnormalities in urinary GAG excretion. MPS patients usually excrete excess GAG in urine and/or have different relative proportions of types of GAG in urine as compared with age-matched normal subjects. Although urinary GAG analyses (both quantitative and qualitative) can suggest the most likely type of MPS, diagnosis must be confirmed by enzyme assay. Multiple assays may be necessary to identify the disease subtype. Correct identification of the MPS type is essential to guide treatment and management decisions.


The pharmacogenetics of CYP2C9 and CYP2C19: ethnic variation and clinical significance.
CYP2C9 and CYP2C19 are important drug metabolizing enzymes and together metabolize about 18% of currently available drugs. Some of the important groups of drugs that are metabolized by them are antihypertensives, hypoglycemics, anticonvulsants, antiulcer drugs etc. Genes encoding these enzymes are polymorphically expressed. Thirty variant alleles for CYP2C9 and 21 for CYP2C19 have been reported. The frequencies of these polymorphic alleles show marked inter-ethnic variation. Several reports have been published showing the clinical importance of this polymorphism. This review summarizes the currently available important information on this topic.


Youth Action Research for Prevention: a multi-level intervention designed to increase efficacy and empowerment among urban youth.
Youth Action Research for Prevention (YARP), a federally funded research and demonstration intervention, utilizes youth empowerment as the cornerstone of a multi-level intervention designed to reduce and/or delay onset of drug and sex risk, while increasing individual and collective efficacy and educational expectations. The intervention, located in Hartford Connecticut, served 114 African-Caribbean and Latino high school youth in a community education setting and a matched comparison group of 202 youth from 2001 to 2004. The strategy used in YARP begins with individuals, forges group identity and cohesion, trains youth as a group to use research to understand their community better (formative community ethnography), and then engages them in using the research for social action at multiple levels in community settings (policy, school-based, parental etc.) Engagement in community activism has, in turn, an effect on individual and collective efficacy and individual behavioral change. This approach is unique insofar as it differs from multilevel interventions that create approaches to attack multiple levels simultaneously. We describe the YARP intervention and employ qualitative and quantitative data from the quasi-experimental evaluation study design to assess the way in which the YARP approach empowered individual youth and groups of youth (youth networks) to engage in social action in their schools, communities and at the policy level, which in turn affected their attitudes and behaviors.


Survey of Toxin⁻Antitoxin Systems in Erwinia amylovora Reveals Insights into Diversity and Functional Specificity.
Toxin⁻antitoxin (TA) systems are diverse genetic modules with demonstrated roles in plasmid stability, stress management, biofilm formation and antibiotic persistence. However, relatively little is known about their functional significance in plant pathogens. In this study we characterize type II and IV TA systems in the economically important plant pathogen Erwinia amylovora. Hidden Markov Model (HMM) and BLAST-based programs were used to predict the identity and distribution of putative TA systems among sequenced genomes of E. amylovora and other plant-associated Erwinia spp. Of six conserved TA systems tested for function from E. amylovora, three (CbtA/CbeA, ParE/RHH and Doc/PhD) were validated as functional. CbtA was toxic to E. amylovora, but not to Escherichia coli. While the E. coli homolog of CbtA elicits the formation of lemon-shaped cells upon overexpression and targets cytoskeletal proteins FtsZ and MreB, E. amylovora CbtA led to cell elongation and did not interact with these cytoskeletal proteins. Phylogenetic analysis revealed that E. amylovora CbtA belongs to a distinct clade from the CbtA of pathogenic E. coli. This study expands the repertoire of experimentally validated TA systems in plant pathogenic bacteria, and suggests that the E. amylovora homolog of CbtA is functionally distinct from that of E. coli.


Animal-based measurements of the severity of mastitis in dairy cows.
A range of clinical parameters were studied to assess their usefulness as objective markers of the severity of clinical mastitis in dairy cows. Cows with moderate clinical mastitis had significantly higher rectal temperatures and heart and respiratory rates than cows with mild clinical mastitis or control cows. The difference in temperature between quarters did not vary significantly between the control cows and the cows with mastitis, but there was a larger difference between quarter temperatures in the cows with moderate mastitis than in the cows with mild mastitis or the control cows. The hock-to-hock distance in the control cows was significantly smaller than in the cows with mild to moderate mastitis, but there was no significant difference in the distance either between the cows with mild mastitis and those with moderate mastitis, or between the cows with mastitis in the front quarters and those with mastitis in the hind quarters. The mechanical threshold to pain of the cows with mild and moderate mastitis was significantly lower than that of the control cows.


Action-specific effects underwater.
Action-specific effects on perception are apparent in terrestrial environments. For example, targets that require more effort to walk, jump, or throw to look farther away than when the targets require less effort. Here, we examined whether action-specific effects would generalize to an underwater environment. Instead, perception might be geometrically precise, rather than action-specific, in an environment that is novel from an evolutionary perspective. We manipulated ease to swim by giving participants swimming flippers or taking them away. Those who estimated distance while wearing the flippers judged underwater targets to be closer than did participants who had taken them off. In addition, participants with better swimming ability judged the targets to be closer than did those with worse swimming ability. These results suggest perceived distance underwater is a function of the perceiver's ability to swim to the targets.


Pulse compression radar reflectometry to measure electron density in plasma with parasitic reflections.
Pulse compression radar reflectometry is used to obtain electron density profile in plasma with parasitic reflections in this article. The pulse compression radar relies on the relation between the temporal width of a pulse and the frequency bandwidth of this pulse: Deltat proportional, variant1Deltaf. So a set of sweep-frequency microwaves within a bandwidth Deltaf can be introduced sequentially into the plasma to obtain the same information as the one obtained by a real pulse. By applying a Fourier transform to the data of reflectivity array in the frequency domain, the temporal response in the time domain is obtained. The limitation of the parasitic reflections on measurement can be eliminated from the temporal response by the method of time gate. This is a prominent advantage when this method is compared to the traditional reflectometry. For this method, an appropriate compromise between the spatial resolution and the electron density resolution is important. Experimental results show that the profile obtained from pulse compression radar reflectometry is similar to that from a double Langmuir probe.


Magnetic Interactions through a Nonconjugated Framework Observed in Back-to-Back Connected Triazinyl-Nitroxyl Biradical Derivatives.
Three hetero-biradical derivatives, with the structure of a back-to-back connected benzotriazinyl and tetramethyl or tetraethylisoindoline N-oxyl sharing a common benzo ring, 3-tert-butyl-1-phenyl-1,4,6,8-tetrahydro-6,6,8,8-tetramethyl-pyrrolo[4,5-g]-1,2,4-benzotriazin-4-yl-7-oxyl (1-tBu), 1,3-diphenyl-1,4,6,8-tetrahydro-6,6,8,8-tetramethyl-pyrrolo[4,5-g]-1,2,4-benzotriazin-4-yl-7-oxyl (1-Ph), and 3-tert-butyl-1-phenyl-1,4,6,8-tetrahydro-6,6,8,8-tetraethyl-pyrrolo[4,5-g]-1,2,4-benzotriazin-4-yl-7-oxyl (2-tBu), were synthesized and characterized by single-crystal X-ray analyses, variable-temperature magnetic susceptibility studies, and DFT calculations. Temperature dependences of the magnetic susceptibilities of 1-tBu, 1-Ph, and 2-tBu exhibit broad maxima at 70, 71, and 43 K, respectively. Although these radical derivatives form a columnar or chained assembly in the solid state, magnetic measurements of diluted samples in the polymer matrices and computational results imply that the magnetic properties of the polycrystalline sample can be explained by a two-spin system with an intramolecular antiferromagnetic interaction. The magnetic behavior can be reproduced by using the Bleaney-Bowers model, with 2J=-80.0 cm-1 for 1-tBu, 2J=-77.1 cm-1 for 1-Ph, and 2J=-48.9 cm-1 for 2-tBu. The moderately strong intramolecular antiferromagnetic interactions can be interpreted by a through-bond interaction through the nonconjugated framework and/or through-space interactions based on molecular orbital theory. The strong distance dependency between the N-O spin site and vinylic carbon atoms indicates that the orbital interaction plays an important role in the intramolecular magnetic interaction. The reduced magnetic interaction in 2-tBu relative to those of 1-tBu and 1-Ph can be attributed to restricted rotation of the tetraethyl group.


Metabolic monitoring and management among clozapine users.
To assess, among clozapine users, the rates of monitoring, presence and treatment of metabolic syndrome and its components. A chart review was conducted of all clozapine users who were followed up in community mental health clinics at two Metro South Health Hospitals over a 1-year period. Metabolic syndrome was diagnosed according to the International Diabetes Federation criteria. We included 251 clozapine users. Only 43.4% (109/251) had data collected for all five metabolic syndrome parameters. Among these people, 45.0% (49/109) met criteria for metabolic syndrome, while 61.2% (30/49) of those with metabolic syndrome were offered appropriate treatments. Correspondence with primary care providers occurred in only 18.7% ( n = 47). Non-pharmacological interventions, such as motivational interviewing and education about healthy lifestyle alternatives, occurred in 49.8% ( n = 125). There is growing awareness of the importance of metabolic monitoring, however, there remain specific gaps in the collaborative work among mental health services, primary care providers and clozapine users, to ensure appropriate physical health interventions.


The use of the index of orthodontic treatment need (IOTN) as a public health tool: a pilot study.
Many attempts have been made in the past to develop indices of malocclusion. The index of orthodontic treatment need was developed in the United Kingdom to assess both dental health and aesthetics, primarily for clinical purposes. This study was designed to assess the use of the index of orthodontic treatment need in dental public health. Dentists in the community dental service were taught how to use the index from a hierarchical training and calibration programme. These dentists then tested the index in the field and reported back their findings. The results indicate that the index of orthodontic treatment need has potential in dental public health but some modifications are suggested. These suggestions will be incorporated into a full study alongside caries prevalence in the future.


Use of Fluorescence Imaging in Combination with Patent Blue Dye versus Patent Blue Dye Alone in Sentinel Lymph Node Biopsy in Breast Cancer.
Near-infrared fluorescence imaging with indocyanine green (ICG) has the potential to improve sentinel lymph node (SLN) mapping in breast cancer. In this clinical trial, we compared the potential value of ICG combined with blue dye with that of blue dye alone for detecting SLNs. Patients undergoing SLN biopsy (SLNB) between November 2010 and November 2013 were included. Up to December 2011, SLNs were detected by using patent blue (PB) alone, and since January 2012, by using PB in combination with ICG. The patients were divided into the following two groups: group A (ICG-PB; n=96) and group B (PB; n=73), and SLN detection parameters were compared between the groups. All patients underwent level I and II axillary dissections after SLNB. In group A, the SLN detection rate was 96.9% (93/96), the accuracy of detection was 98.9% (92/93), and the false-negative rate (FNR) was 3.4% (1/29). In group B, the SLN detection rate was 84.9% (62/73), the accuracy of detection was 96.8% (60/62), and the FNR was 11.1% (2/18). The ICG-PB group showed significantly superior results compared to the PB group for SLN detection (p=0.005) and a greatly improved FNR. The combined fluorescence and blue dye-based tracer technique was superior to the use of blue dye alone for identifying SLNs, and for predicting axillary lymph node status in patients with breast cancer; in addition, the combined technique had reduced false-negative results.


Understanding burnout and moral distress to build resilience: a qualitative study of an interprofessional intensive care unit team.
The purpose of this study was to explore personal and organizational factors that contribute to burnout and moral distress in a Canadian academic intensive care unit (ICU) healthcare team. Both of these issues have a significant impact on healthcare providers, their families, and the quality of patient care. These themes will be used to design interventions to build team resilience. This is a qualitative study using focus groups to elicit a better understanding of stakeholder perspectives on burnout and moral distress in the ICU team environment. Thematic analysis of transcripts from focus groups with registered intensive care nurses (RNs), respiratory therapists (RTs), and physicians (MDs) considered causes of burnout and moral distress, its impact, coping strategies, as well as suggestions to build resilience. Six focus groups, each with four to eight participants, were conducted. A total of 35 participants (six MDs, 21 RNs, and eight RTs) represented 43% of the MDs, 18.8% of the RNs, and 20.0% of the RTs. Themes were concordant between the professions and included: 1) organizational issues, 2) exposure to high-intensity situations, and 3) poor team experiences. Participants reported negative impacts on emotional and physical well-being, family dynamics, and patient care. Suggestions to build resilience were categorized into the three main themes: organizational issues, exposure to high intensity situations, and poor team experiences. Intensive care unit team members described their experiences with moral distress and burnout, and suggested ways to build resilience in the workplace. Experiences and suggestions were similar between the interdisciplinary teams.


Reidentification of larval interneurons in the pupal stage of the tobacco hornworm, Manduca sexta.
The abdominal prolegs are the primary locomotory appendages of Manduca sexta larvae. After the prolegs are lost at pupation, some of the proleg motoneurons die while the survivors are respecified to carry out different functions in the adult moth. As a first step toward investigating the process of functional respecification at the synaptic level, we searched for larval interneurons that affected the activity of proleg motoneurons, and followed these interneurons into the pupal stage. Interneurons were judged to be individually identifiable based on their effects on proleg motoneuron activity and their anatomical features. Seven larval interneurons were identified and placed in five physiological classes based on their effects on proleg motoneurons: ipsilateral excitors, contralateral excitors, ipsilateral inhibitors, contralateral inhibitors, and bilateral inhibitor-excitors. Four of the larval interneurons produced apparently monosynaptic postsynaptic potentials in proleg motoneuron. Of the five larval interneurons that were reidentified in the early pupal stage, two showed minor but consistent structural modifications from the larval stage. Interneurons that produced unitary postsynaptic potentials in larval motoneurons continued to do so in pupal motoneurons. These studies demonstrate that individually identified interneurons can be followed through the larval-pupal transformation, during the initial stages of motoneuron respecification.


The generation and biological activity of a long-lasting recombinant human interferon-λ1.
The previously generated recombinant human (rh) interferon (IFN)-λ1 protein has a short half-life, and this feature makes it challenging to conduct studies on potential clinical applications for rhIFN-λ1. In an attempt to overcome this difficulty, we constructed a 'long-life' version of rhIFN-λ1. This modified rhIFN-λ1, named rhIFN-λ1-CTPON, has a human chorionic gonadotropin β subunit carboxyl-terminal peptide (CTP) and an N-glycosylation sequence linked to its C-terminus. We confirmed the sequence of rhIFN-λ1-CTPON by mass spectrometry and then measured its biological activities. The results show that rhIFN-λ1-CTPON had antiviral activity and anti-proliferation activity in vitro that were similar to those of rhIFN-λ1 and that it similarly promoted natural killer cell cytotoxicity. Notably, the in vivo half-life of rhIFN-λ1-CTPON was determined to be 3-fold higher than that of rhIFN-λ1. We also assessed the anti-hepatitis B virus activity of rhIFN-λ1-CTPON; it was able to inhibit the production of the antigens HBs-Ag and HBe-Ag and induce antiviral gene expression. In conclusion, rhIFN-λ1-CTPON has a longer half-life than rhIFN-λ1 and has similar biological activities, so rhIFN-λ1-CTPON is an appropriate substitute for rhIFN-λ1 in the further study of potential clinical applications for rhIFN- λ1.


Growth and cellular proliferation of ovine corpora lutea throughout the estrous cycle.
This study was conducted to determine the rates of growth and cellular proliferation of ovine corpora lutea (CL) throughout the estrous cycle. To determine the cellular labeling index (LI), ewes received an iv injection of bromodeoxyuridine (BrdU) 1 h before death on days 2, 4, 8, 12, or 15 (day 0 = estrus; n = 6-12 ewes/day). At death, CL were weighed, and samples of each were fixed in Carnoy's solution or frozen until analyzed for DNA, protein, and progesterone contents. Nuclear incorporation of BrdU was determined in paraffin-embedded tissue sections by using a primary antibody against BrdU and a fluorescent (fluorescein isothiocyanate-labeled) secondary antibody, and sections were counterstained with propidium iodide (a nuclear stain). The labeling index (BrdU-labeled nuclei as a proportion of propidium iodide-labeled nuclei) of each CL was determined by using dual channel interactive laser cytometry and image analysis. Moreover, BrdU and 3 beta-hydroxysteroid dehydrogenase (a marker for steroidogenic cells) or BrdU and factor VIII (a marker for endothelial cells) were immunolocalized in tissue sections by using double immunohistochemical or dual immunofluorescent staining, respectively. Results demonstrated that cellular proliferation was greatest (LI, 34.1 +/- 2.1%) on day 2 and decreased (P < 0.01) through day 15 (LI, 0.7 +/- 0.1%) of the estrous cycle. The results of the immunohistochemical studies provide evidence that both parenchymal (steroidogenic) and nonparenchymal (e.g. endothelial, fibroblastic) luteal cells proliferated throughout the ovine estrous cycle. Conversely, from days 2-12 of the estrous cycle, fresh weight and DNA content of CL increased linearly (P < 0.01; 8- and 10-fold, respectively), then decreased (P < 0.02) from days 12-15. Ratios of protein/DNA on days 2, 4, and 8 were similar and were greater (P < 0.02) than those on days 12 and 15, which also were similar. These data demonstrate that growth of the ovine CL is extremely rapid, linear from days 2-12, and primarily due to hyperplasia. In addition, the high rate of cellular proliferation is associated primarily with nonsteroidogenic cells, a large proportion of which appear to be endothelial cells. Data such as these will enable us to determine the factors that are important in regulating luteal growth and development in normal and pathological conditions.


Fluorometric method for the simultaneous quantitation of differently-sized nanoparticles in rodent tissue.
The oral absorption and systemic translocation of particulate matter via the gastrointestinal tract has been shown by a number of laboratories using a wide variety of particles in different animal species. While there is debate on the magnitude of particle intestinal translocation, which is encumbered by the differing experimental protocols, particularly the method of quantitation of absorbed material, few have sought to examine the pharmacokinetic aspects of particle absorption. We describe in this communication the development of a simple and a rapid fluorometric assay of quantifying tissue-laden fluorescent nanoparticles that is able to isolate, detect and quantify the presence of two or more particle populations differing both in their size and fluorescent label. Six types of polystyrene nanoparticles incorporating different fluorescent markers were spiked in whole livers. The fluorophores were extracted using our previously developed method of freeze-drying the tissue and using chloroform as the extractive solvent. Only two types of particle populations, orange-labelled 40 nm and Fluoroscein-emitting 500 nm nanoparticles, were sufficiently recoverable and provided a high signal-to-noise ratio for further work. The amount of tissue and type of biological tissue type also impacted on the nanoparticle recovery and detection, reflecting, perhaps, the quenching effects of interacting tissue-derived molecules. In addition, the results also indicate that the use of nanoparticles incorporating fluorescent dyes that have emission over 500 nm overcome the tissue interfering autofluorescence for low doses of nanoparticles. The use of this fluorometric method has several advantages compared with other modes of quantitation in that it is rapid, non-radioactive and the marker is non-leaching. More importantly, it allows the simultaneous detection of multiple fluorophores such that two or more different fluorescent particle populations can be detected in the same sample. This may enable the uncharted area of pharmacokinetic parameters, such as the impedance, augmentation or site of gut uptake of differently sized particles to be studied.


Translocation of PKN from the cytosol to the nucleus induced by stresses.
Effects of environmental stresses on the subcellular localization of PKN were investigated in NIH 3T3, BALB/c 3T3, and Rat-1 cells. The immunofluorescence of PKN resided prominently in the cytoplasmic region in nonstressed cells. When these cells were treated at 42 degrees C, there was a time-dependent decrease of the immunofluorescence of PKN in the cytoplasmic region that correlated with an increase within the nucleus as observed by confocal microscope. After incubation at 37 degrees C following beat shock, the immunofluorescence of PKN returned to the perinuclear and cytoplasmic regions from the nucleus. The nuclear translocation of PKN by heat shock was supported by the biochemical subcellular fractionation and immunoblotting. The nuclear localization of PKN was also observed when the cells were exposed to other stresses such as sodium arsenite and serum starvation. These results raise the possibility that there is a pathway mediating stress signals from the cytosol to the nucleus through PKN.


Synthesis of Gallic-Acid-1-Phenyl-1H-[1,2,3]Triazol-4-yl Methyl Esters as Effective Antioxidants.
Using a click chemistry approach, a series of gallic-acid-1-phenyl-1H-[1,2,3]triazol-4-ylmethyl esters was synthesized to develop more effective antioxidants. The results of DPPH screening indicate that few of the synthesized analogs display better antioxidant effect compared to the standards. Among all, compounds, 9 and 20 displayed highest DPPH radical scavenging effect with IC50 values as low as 6.4±0.2 and 7.9±0.4 µM respectively, compared to the standard ascorbic acid (IC50=12±0.8 µM) and gallic acid (IC50=9.0±0.6 µM). Compound 10 also displayed a potent antioxidant effect with IC50 of 10.80±0.4 µM. This study provides an important aspect with regard to the use of these gallic-acid based synthetic antioxidants in food industry as dietary supplements.


The major histocompatibility complex and genetic control of antibody response to synthetic antigens in chickens.
The genetic association of the B-F/B-L region of the chicken major histocompatibility complex (MHC) with the antibody responses to the hapten 2,4-dinitrophenol (DNP) conjugated to human gamma globulin and to the synthetic antigens, GAT and (T,G)-A-L were investigated using five inbred lines of White Leghorn chickens. Two of the five inbred lines of chickens J1 and M1, were found to be high responders to DNP and (T,G)-A-L, whilst L1, M4 and N1 chickens were found to be relatively low responders to both antigens. Chickens from all five inbred lines were low responders to GAT. Matings between the inbred lines. J1, M1 and N(1), showed that the magnitude of the antibody response to (T,G)-A-L in backcross chickens was regulated by a dominant gene or set of genes linked to the MHC. High antibody responsiveness to (T,G)-A-L was inherited as a dominant trait linked to the B113 and the B15 MHC alleles of the J1 and M1 inbred lines respectively. The similarity of the anti-(T,G)-A-L responses in chickens from the three B113 homozygous inbred lines L1, M4 and N1, also suggests that control of this response was linked to the MHC. The magnitude of the antibody response to DNP in backcross chickens from matings between the inbred lines N1 and J1 or M1 was also found to be regulated by a gene or set of genes linked to the MHC. Again, high antibody responsiveness to DNP was inherited as a dominant trait linked to the B113 and the B15 MHC alleles of the J1 and M1 inbred lines respectively.


A Physics-Based Deep Learning Approach to Shadow Invariant Representations of Hyperspectral Images.
This paper proposes the Relit Spectral Angle-Stacked Autoencoder, a novel unsupervised feature learning approach for mapping pixel reflectances to illumination invariant encodings. This work extends the Spectral Angle-Stacked Autoencoder so that it can learn a shadow-invariant mapping. The method is inspired by a deep learning technique, Denoising Autoencoders, with the incorporation of a physics-based model for illumination such that the algorithm learns a shadow invariant mapping without the need for any labelled training data, additional sensors, a priori knowledge of the scene or the assumption of Planckian illumination. The method is evaluated using datasets captured from several different cameras, with experiments to demonstrate the illumination invariance of the features and how they can be used practically to improve the performance of high-level perception algorithms that operate on images acquired outdoors.


The effect of cryopreservation on the survival and MHC antigen expression of murine islet allografts.
Cryopreservation is an effective method of islet storage that can facilitate clinical trials of islet transplantation. In the present study we examined the effect of cryopreservation on the survival of islet allografts and the quantity of islet MHC antigen expression. Islets isolated from CBA/J (H-2k) mice were transplanted into streptozotocin-induced diabetic BALB/c (H-2d) mice treated with or without antilymphocyte serum (ALS). Frozen/thawed (F/T) grafts were cooled slowly to -40 degrees C, stored at -196 degrees C, and thawed rapidly. Fresh and F/T isograft controls reversed diabetes promptly and maintained normoglycemia > 100 days. Allografts of fresh and F/T islets induced normoglycemia initially, but graft failure ensued at 18.2 +/- 1.5 and 16.4 +/- 1.9 days, respectively. ALS treatment prolonged allograft survival significantly to 35.3 +/- 3.9 and 37.5 +/- 6.3 days for fresh and F/T islets, respectively. Following cryopreservation, the quantity of class I antigen expression was reduced by 40%, while the quantity of class II expression was variable. These data indicate that murine islet MHC class I expression is reduced after cryopreservation. This decrease was not associated with altered survival of allogeneic grafts.


MiR-199a-3p/5p participated in TGF-β and EGF induced EMT by targeting DUSP5/MAP3K11 in pterygium.
Recently, it has been reported that miRNA is involved in pterygium, however the exact underlying mechanism in pterygium is unrevealed and require further investigation. The differential expression of miRNA in pterygium was profiled using microarray and validated with quantitative real-time polymerase chain reaction (qRT-PCR). Human conjunctival epithelial cells (HCEs) were cultured and treated with transforming growth factor β (TGF-β) and epidermal growth factor (EGF) and transfected with miR-199a-3p/5p mimic and inhibitor. Markers of epithelial-mesenchymal transition (EMT) in HCEs were detected using western blot and immunohistochemistry. Cell migration ability was determined using wound healing and transwell assay, while apoptosis was determined by flow cytometry. The target genes of miR-199a were confirmed by the dual-luciferase reporter assay. TGF-β and EGF could induced EMT in HCEs and increase miR-199a-3p/5p but suppress target genes, DUSP5 and MAP3K11. With the occurrence of EMT, cell migration ability was enhanced, and apoptosis was impeded. Promoting miR-199a-3p/5p expression could induce EMT in HCEs without TGF-β and EGF, while suppressing miR-199a-3p/5p could inhibit EMT in TGF-β and EGF induced HCEs. In a word, TGF-β and EGF induced EMT could be regulated with miR-199a-3p/5p-DUSP5/MAP3K11 axes. The validated results in tissues showed that, compared with control conjunctival tissues, miR-199a-3p/5p were more overexpressed in pterygium, while DUSP5/MAP3K11 were lower expressed. In addition, bioinformatics analysis indicated the miR-199a-3p/5p-DUSP5/MAP3K11 was belong to MAPK signalling pathway. TGF-β and EGF induce EMT of HCEs through miR-199a-3p/5p-DUSP5/MAP3K11 axes, which explains the pathogenesis of EMT in pterygium and may provide new targets for pterygium prevention and therapy.


Intratunical injection of autologous adipose stromal vascular fraction reduces collagen III expression in a rat model of chronic penile fibrosis.
Previous studies have shown that the injection of adipose stem cells and stromal vascular fraction(SVF) into the tunica albuginea (TA) during the inflammatory phase in a rat model of Peyronie's disease(PD) prevented the development of TA fibrosis. Our aim was to investigate whether local injection of SVF can reduce established fibrosis in a rat model of chronic phase of PD. Eighteen-male 12-wk-old Sprague-Dawley rats were divided in three equal groups: sham, PD without treatment (PD) and PD treated with SVF(PD-SVF). Sham rats underwent 2 injections of vehicle into the TA one month apart. PD rats underwent TGF-β1 injection and injection of vehicle one month later. PD-SVF rats underwent TGF-β1 injection followed by SVF (1-million cells) one month later. One month after the last treatment, the animals, n = 6 rats per group, underwent measurement of intracorporal and mean arterial pressure during electrostimulation of the cavernous nerve. Following euthanasia, penises were harvested for in-vitro study. Erectile function was not statistically significantly different between groups. PD animals developed subtunical areas of fibrosis and elastosis with upregulation of collagen III protein. These fibrotic changes were reversed after injection of SVF. We provide evidence that local injection of SVF reverses TA fibrosis in a rat model of chronic phase of PD.


Cation-controlled crystal growth of silver stearate: cryo-TEM investigation of lithium vs sodium stearate.
Cryo-TEM, SAXS, and light microscopy techniques were used to probe the morphology and kinetics of silver stearate self-assembly and crystallization from the reaction of silver nitrate with lithium stearate. Unlike the reaction of sodium stearate with silver nitrate, which proceeds via micelle aggregation, the lithium stearate forms vesicles that drastically change the reaction kinetics of the silver stearate nucleation and self-assembly process. In addition, even with excess silver nitrate present, only about 80% of the lithium stearate can be converted to silver stearate. The presence of the residual lithium stearate inhibits the silver stearate crystal growth process. Consequently, no silver stearate micelle aggregates of any significant size form, unlike the system utilizing sodium stearate. Instead, significantly smaller silver stearate crystals result from lithium stearate compared to the silver stearate crystals from sodium stearate and provide an opportunity to further control silver stearate self-assembly and crystal growth.


[Magnesium supplementation to patients with type II diabetes].
The purpose of this study was to evaluate Mg status by nuclear magnetic resonance spectroscopy in a group of well-regulated non-insulin-dependent diabetic (NIDDM) patients without angiopathy. Furthermore, to investigate the effect of Mg supplementation on markers of diabetic control, hemostatic function, platelet reactivity and endothelial function in the same patient population. A double-blinded, placebo-controlled and randomized crossover study was carried out, with two 8-weeks treatment periods (360 mg Mg/day) separated by a 4-weeks wash-out period. 11 well-regulated NIDDM patients participated in the study. Eight weeks of Mg supplementation significantly raised the level of free intracellular Mg in the diabetic patients (157.35 +/- 16.53 vs. 197.49 +/- 27.60 microM; p < 0.01). No changes were observed neither in plasma level of von Willebrand factor antigen, fibrinogen and fibronectin nor in platelet release of thromboxane B2 (TxB2). Similarly, markers of diabetic regulation, HbA1c and fructosamine, showed no significant changes. These results suggest that even well regulated NIDDM patients have marked Mg deficiency. Restoring this deficiency had no effect on diabetic control, markers of platelet reactivity, hemostatic function and endothelial function.


Inadequate food intake among adults living with HIV.
CONTEXT AND OBJECTIVE The number of people living with HIV (PLHIV) in Brazil is between 600,000 and 890,000. Assessing the diet is important in planning healthcare actions and improving PLHIV's quality of life. This study aimed to estimate the prevalence of inappropriate protein, total fat, saturated fat, carbohydrate, fiber, sodium, calcium and cholesterol intake among PVHIV on highly-active antiretroviral therapy (HAART). DESIGN AND SETTING Cross-sectional study in nine Specialized STD/AIDS Healthcare Centers in São Paulo. METHODS Men and women aged 20 to 59 years, on HAART for at least three months, were included. Nutrient intake was assessed using 24-hour food recall applied in person and repeated among 30% of the population by telephone. The between and within-person variances were corrected. RESULTS 507 individuals were evaluated: 58% male, mean age 41.7 years (standard deviation, SD = 7.8). The mean time since HIV diagnosis was 6.6 years (SD = 4.1), and since HAART onset, 5.1 years (SD = 3.3). More than 20% of the population presented intake above the recommendations for saturated fat, cholesterol and/or sodium, and below the recommendations for fiber. The recommended maximum tolerable sodium level was exceeded by 99% of the sample, and 86% of men and 94% of women did not reach the daily recommendations for calcium. Protein, carbohydrate and total fat intakes were adequate for the majority of the population. CONCLUSIONS A significant portion of the population presented inappropriate intake of saturated fat, sodium, fiber and calcium. Interventions aimed at improving PLHIV's dietary quality are needed.


The HevCaLP protein mediates binding specificity of the Cry1A class of Bacillus thuringiensis toxins in Heliothis virescens.
Retrotransposon-mediated disruption of the BtR-4 gene encoding the Heliothis virescens cadherin-like protein (HevCaLP) is linked to high levels of resistance in the YHD2 strain to Cry1Ac toxin from Bacillus thuringiensis. This suggests that HevCaLP functions as a Cry1Ac toxin receptor on the surface of midgut cells in susceptible larvae and that the BtR-4 gene disruption eliminates this protein in resistant larvae. However, Cry1Ac toxin binding to HevCaLP is yet to be reported. We used the polymerase chain reaction and immunoblotting as tools to discriminate between individual H. virescens larval midguts from susceptible (YDK) and resistant (CXC, KCBhyb, and YHD2-B) strains according to their BtR-4 gene disruption genotype and phenotype. This approach allowed us to test the correlation between BtR-4 gene disruption, lack of HevCaLP, and altered Cry1A toxin binding. Toxin-binding assays using brush border membrane vesicles revealed that a wild-type BtR-4 allele is necessary for HevCaLP production and Cry1Aa toxin binding, while most of Cry1Ab and Cry1Ac binding was independent of the BtR-4 genotype. Moreover, toxin competition experiments show that KCBhyb midguts lacking HevCaLP are more similar to midguts of the original YHD2 strain than to the current YHD2-B strain. This resolves discrepancies in published studies of Cry1A binding in YHD2 and supports our earlier suggestion that a separate genetic change occurred in YHD2 after appearance of the cadherin disruption, conferring even higher resistance in the resulting YHD2-B strain as well as a large reduction in Cry1Ab and Cry1Ac binding.


Spontaneous resolution of an isolated cervical anterior spinal artery aneurysm after subarachnoid hemorrhage.
Isolated cervical anterior spinal artery aneurysms are extremely rare. Subarachnoid hemorrhage (SAH) secondary to such lesions have been described only in six cases to the best of our knowledge. We describe an unusual clinical picture of SAH due to rupture of anterior spinal artery aneurysm in a patient with previous normal angiogram. Due to the location of the aneurysm and clinical status of the patient, conservative management was proposed, and she was discharged to further follow-up. Monthly routine angiograms revealed resolution of the aneurysm 90 days after bleeding, which was highly suggestive of vascular dissection. We highlight the need to consider these aneurysms in the differential diagnosis of SAH, especially when occurring in the posterior fossa and when angiography findings are inconclusive.


Oral absorption of anti-AIDS nucleoside analogues. 1. Intestinal transport of didanosine in rat and rabbit preparations.
The intestinal transport of didanosine (ddl), a nucleoside analog used in the treatment of human immunodeficiency virus (HIV) infection, was characterized using in situ and in vitro techniques. The zero-trans uptake of ddl in rat intestinal brush border membrane vesicles (BBMV) was linear over the range of 1 microM to 50 mM, ruling out a significant carrier-mediated absorption component. The lack of carrier-mediated transport was confirmed in a second species (rabbit). In order to quantitate the convective (Pconv) and diffusive (Pdiff) components of ddl intestinal permeability, the steady state wall permeability (P*w) was determined using an established perfusion technique in rats. Even though baseline P*w (pH 6.5, 290 mosm/kg, no modulator) and fluid absorption results were similar to those of furosemide, the ratios (ddl:furosemide) of Pdiff and phi, the sieving coefficient, were 0.31:1 and 1.70:1, respectively, demonstrating that ddl's Pdiff is low and Pconv is high relative to furosemide's, suggesting significant paracellular absorption of ddl. The apparent diffusive absorptive clearances (P'app) of ddl and furosemide were determined in BBMV, which lack functional tight junctions, and the ratios (ddl:furosemide) of P'app in rat and rabbit were 0.23:1 and 0.24:1, respectively. The BBMV results demonstrate that the majority of ddl intestinal transport does not occur by passive membrane diffusion, confirming the single pass intestinal perfusion (SPIP) findings. The results of these studies suggest that ddl is transported by nonfacilitated membrane and paracellular diffusion with paracellular transport being responsible for the majority of ddl absorption from the intestine.


A comparison of prediction equations for estimating glomerular filtration rate in Chinese potential living kidney donors.
Accurate measurement of donor renal function has important long-term implications for both donors and recipients. In clinical practice, renal function may be estimated by using 24-h urinary creatinine clearance (urine-CrCl) and various specifically derived prediction equations. We assessed the suitability of urine-CrCl and prediction equations for evaluating Chinese kidney donors. A total of 224 donors who had undergone (99m)Tc-diethylenetriaminepentaacetic acid glomerular filtration rate (GFR) estimation were enrolled. We evaluated the performance of the Cockcroft-Gault equation (CG-CrCl), the CG-CrCl equation corrected for GFR (CG-GFR), the isotope dilution mass spectrometry traceable modification of diet in renal disease (IDMS-MDRD) study equation, the new MDRD study equation for Chinese (C-MDRD 1), the modified MDRD study equation with a Chinese coefficient (C-MDRD 2), the modified IDMS-MDRD study equation with a Japanese coefficient (J-MDRD), and urine-CrCl in predicting GFR before donation. Of 224 donors selected, 68 were male and 156 were female. Of all the prediction equations, C-MDRD 1 was the least scattered and most precise. However, predictive performance was poor for all the equations. The performance of urine-CrCl and all the equations was disappointing, and even the best performing equation C-MDRD 1 was unacceptable. Considering the potential risk of living kidney donation, other more accurate methods of GFR measurement should be used in clinical practice.


A comparison of the effect of five phenothiazines on hepatic CYP isoenzymes in rats.
We examined the influence of five phenothiazine derivatives on the activity and the expression of hepatic cytochrome P450 (CYP) 1A, 2B, 2C6, 2C11, 2E1 and 3A2 in male Sprague-Dawley rats. Twenty mg/kg of phenothiazine, chlorpromazine, and thioridazine, or 1 mg/kg of trifluoperazine and perphenazine was administered intraperitoneally for 4 consecutive days. Phenothiazine increased the total CYP content and induced CYP1A, CYP2B, and CYP3A. Western blot analysis showed marked induction of CYP1A1 by phenothiazine. Chlorpromazine induced CYP2B, and CYP3A without a significant increase in the CYP content. Thioridazine decreased the total CYP content and reduced CYP2C11, CYP2E1, and CYP3A. Neither trifluoperazine nor perphenazine significantly altered the catalytic activity or the protein level of any enzyme examined. These results suggest that the antipsychotic phenothiazine derivatives examined here hardly affect CYP1A at the pharmacological doses, while phenothiazine does induce it. Although a relatively short period and small dose of administration might counteract the influence, trifluoperazine and perphenazine have less effect on CYP subfamilies than chlorpromazine or thioridazine.


Immediate placement of single implants with or without immediate provisionalization in the maxillary aesthetic region: A 5-year comparative study.
To compare marginal bone level changes around immediately placed and immediately provisionalized implants with immediately placed and delayed provisionalized implants in the aesthetic region after five years of function. Forty patients with a failing tooth in the maxillary anterior region were randomly assigned immediate implant placement with immediate (Group A: n = 20) or delayed (Group B: n = 20) provisionalization. Definitive crown placement occurred three months after provisionalization. The primary outcomes were changes in marginal bone level. In addition, survival rates, buccal bone thickness, soft peri-implant tissues, aesthetics and patient-reported outcomes were assessed. After 5 years, the mean mesial and distal marginal bone level changes were 0.71 ± 0.68 mm and 0.71 ± 0.71 mm, respectively, in group A and 0.49 ± 0.52 mm and 0.54 ± 0.64 mm, respectively, in group B; the difference between the groups was not significant (p = .305 and p = .477, respectively). Implant and restoration survivals were 100%. No clinically relevant differences in buccal bone thickness or in mid-facial peri-implant mucosal level, aesthetic and patient outcomes were observed. The mean marginal bone level changes following immediate implant placement and provisionalization were comparable with immediate implant placement and delayed provisionalization. (www.isrctn.com: ISRCTN57251089 and www.trialregister.nl: NL8255).


[A case of slowly progressive type 1 diabetes mellitus developing myeloperoxidase-specific anti-neutrophil cytoplasmic antibody-associated vasculitis with hypertrophic pachymeningitis manifesting as multiple cranial nerve palsy].
We report a 63-year-old man with a 35-year history of slowly progressive type 1 diabetes mellitus (SPIDDM), complicated with myeloperoxidase-specific anti-neutrophil cytoplasmic antibody (MPO-ANCA)-associated vasculitis presenting alveolar hemorrhage and pachymeningitis. The patient was first diagnosed as having DM at age of 28 years old and deteriorated secretion of insulin and the typical clinical course led us to the diagnosis of SPIDDM. When he was 58 years old, he suffered from fever, headache, and alveolar hemorrhage. He was diagnosed as having MPO-ANCA associated vasculitis based on a high titer of MPO-ANCA and histological findings of lung biopsy. Treatment with steroid pulse therapy, followed by oral prednisolone and oral cyclophosohamide, resulted in clinical improvement. Five years later, he complained of double vision. A gadolinium-enhanced magnetic resonance imaging (MRI) study of the brain showed normal. Two months later, he developed right cranial nerve V~XII palsy. A second MRI study revealed thickening of the right temporal region and cerebellar dura mater, leading us to the diagnosis of hypertrophic pachymeningitis. He responded well to oral prednisolone (50 mg/day) and intravenous cyclophosohamide (500 mg). This is the first case report of SPIDDM complicated with MPO-ANCA-associated vasculitis, manifesting as alveolar hemorrhage and hypertrophic pachymeningitis.


Latent evolutionary potentials under the neutral mutational drift of an enzyme.
Biological systems exhibit mutational robustness, or neutrality, whereby the impact of mutations is minimized. Does neutrality hamper their ability to adapt in the face of changing environments? We monitored changes in genotype and phenotype that occur within a neutral mutational network of an enzyme, experimentally and computationally (see accompanying article). Using the enzyme PON1 as a model, we performed random mutagenesis and purifying selection to purge deleterious mutations. We characterized approximately 300 variants that are apparently neutral, or close to neutral, with respect to PON1's levels of expression and native lactonase activity. Their activities with promiscuous substrates and ligands indicated significant changes in adaptive potentials. Almost half of the variants exhibited changes in promiscuous activities, specificities, or inhibition, and several of these were found to be one or two mutations, closer to potentially new phenotypes: aryl esterase, thiolactonase, phosphotriesterase, or drug resistance. This empirical measure of phenotypic changes under neutrality supports the notion that sequence changes that are neutral, i.e., non-adaptive, in a current context can facilitate adaptation under changing circumstances, by both expanding the activity range of existing enzymes and thus providing an immediate advantage, and by reducing the number of mutations required for divergence of new functions.


[Antitumor activity and structure-activity relationship of seven lanostane-type triterpenes from Fomitopsis pinicola and F. officinalis].
Seven lanostane-type triterpenes including fomitopsin C(1),3-keto-dehydrosulfurenic acid(2),dehydroeburiconic acid(3),3-acetyloxylanosta-8, 24-dien-21-oic acid(4),pinicolic acid A(5),trametenolic acid B(6),and eburicoic acid(7),were isolated from the fruitbodies of Fomitopsis pinicola and F. officinalis. In vitro assay, all compounds were evaluated against MCF-7, HeLa, HepG2 and A549 cells lines using the MTT assay and the structure-activity relationship of antitumor activity was discussed. The results showed that the seven compounds were more sensitive to MCF-7 cells.The IC₅₀ value for MCF-7 was 2<5<4<1<3<6<7. H22 tumor mouse model was used to assay compounds 2, 3, 4 and 5 in vivo. Compounds 2 and 4 had obvious effect and the necrosis area and measurement were positively correlated. The results showed that compounds 2, 4 and 5 had significant antitumor activities at a dose of 20 mg•L⁻¹ with 65.31%, 56.71%, 58.72% suppression, respectively, approaching to CTX group with 69.19% suppression in subcutaneous H22-implanted mice.The results showed that these compounds had significant against the expression of VEGF, cytokines IL-4 and IFN-γ tumor, additionally, the structure-activity relationship of lanostane-type triterpenes indicated that the acetoxyl or carbonyl at C-3 and hydroxy at C-15 can enhance the antitumor activity.


Facilitated recruitment of mesenchymal stromal cells by bone marrow concentrate and platelet rich plasma.
Biologics containing growth factors are frequently used to enhance healing after musculoskeletal injuries. One mechanism of action is thought to be though the ability of biologics to induce homing and migration of endogenous mesenchymal stromal cells (MSCs) to a target tissue. However, the ability of biologics to stimulate chemotaxis (directed migration of cells) and chemokinesis (increase rate of cell migration) of MSCs is unknown. The aim of this study was to directly compare the ability of biologics including platelet rich plasma (PRP) and bone marrow concentrate (BMC) to induce MSC migration. The hypothesis was that leukocyte-low platelet rich plasma (Llo PRP) would induce migration to a greater extent than leukocyte-high platelet rich plasma (Lhi PRP) or BMC. Bone marrow-derived MSCs were isolated from 8 horses. Migration of MSCs toward a biologic (BMC, Llo PRP, and Lhi PRP) or the positive control platelet derived growth factor (PDGF) was continuously traced and measured for 24hrs using time-lapse microscopy and a microfluidics device. Cell migration, chemotaxis and chemokinesis were determined by measurements of displacement, number of cells migrated, and cell flux. All biologics resulted in a significantly greater percentage of MSCs migrated compared to the positive control (PDGF). MSCs migrated further toward BMC compared to Llo PRP. Cell migration, measured as cell flux, was greater toward BMC and Lhi PRP than Llo PRP. The biologics BMC and Lhi PRP elicit greater chemotaxis and chemokinesis of MSCs than Llo PRP. However, all biologics recruited the same number of MSCs suggesting that differences in other regenerative effects, such as growth factor concentration, between biologics should be strongly considered when choosing a biologic for treatment of musculoskeletal injuries. The results of this study have the potential to reduce the need, risks, and costs associated with MSC culture and delivery.


Coxiella burnetii Sterol-Modifying Protein Stmp1 Regulates Cholesterol in the Intracellular Niche.
Coxiella burnetii replicates in a phagolysosome-like vacuole called the Coxiella-containing vacuole (CCV). While host cholesterol readily traffics to the CCV, cholesterol accumulation leads to CCV acidification and bacterial death. Thus, bacterial regulation of CCV cholesterol content is essential for Coxiella pathogenesis. Coxiella expresses a sterol-modifying protein, Stmp1, that may function to lower CCV cholesterol through enzymatic modification. Using an Stmp1 knockout (Δstmp1), we determined that Stmp1 is not essential for axenic growth. Inside host cells, however, Δstmp1 mutant bacteria form smaller CCVs which accumulate cholesterol, preferentially fuse with lysosomes, and become more acidic, correlating with a significant growth defect. However, in cholesterol-free cells, Δstmp1 mutant bacteria grow similarly to wild-type bacteria but are hypersensitive to cholesterol supplementation. To better understand the underlying mechanism behind the Δstmp1 mutant phenotype, we performed sterol profiling. Surprisingly, we found that Δstmp1 mutant-infected macrophages accumulated the potent cholesterol homeostasis regulator 25-hydroxycholesterol (25-HC). We next determined whether dysregulated 25-HC alters Coxiella infection by treating wild-type Coxiella-infected cells with 25-HC. Similar to the Δstmp1 mutant phenotype, 25-HC increased CCV proteolytic activity and inhibited bacterial growth. Collectively, these data indicate that Stmp1 alters host cholesterol metabolism and is essential to establish a mature CCV which supports Coxiella growth. IMPORTANCE Coxiella burnetii is the causative agent of human Q fever, an emerging infectious disease and significant cause of culture-negative endocarditis. Acute infections are often undiagnosed, there are no licensed vaccines in the United States, and chronic Q fever requires a prolonged antibiotic treatment. Therefore, new treatment and preventive options are critically needed. Coxiella is an obligate intracellular bacterium that replicates within a large acidic phagolysosome-like compartment, the Coxiella-containing vacuole (CCV). We previously discovered that cholesterol accumulation in the CCV increases its acidification, leading to bacterial death. Therefore, in order to survive in this harsh environment, Coxiella likely regulates CCV cholesterol levels. Here, we found that Coxiella sterol modifying protein (Stmp1) facilitates bacterial growth by reducing CCV cholesterol and host cell 25-hydroxycholesterol (25-HC) levels, which prevents excessive CCV fusion with host lysosomes and CCV acidification. This study establishes that Stmp1-mediated regulation of host cholesterol homeostasis is essential for Coxiella intracellular survival.


GPR39: a Zn(2+)-activated G protein-coupled receptor that regulates pancreatic, gastrointestinal and neuronal functions.
GPR39 is a vertebrate G protein-coupled receptor related to the ghrelin/neurotensin receptor subfamily. The receptor is expressed in a range of tissues including the pancreas, gut/gastrointestinal tract, liver, kidney and in some regions of the brain. GPR39 was initially thought to be the cognitive receptor for the peptide hormone, obestatin. However, subsequent in vitro studies have failed to demonstrate binding of this peptide to the receptor. Zn(2+) has been shown to be a potent stimulator of GPR39 activity via the Gα(q), Gα(12/13) and Gα(s) pathways. The potency and specificity of Zn(2+) in activating GPR39 suggest it to be a physiologically important agonist. GPR39 is now emerging as an important transducer of autocrine and paracrine Zn(2+) signals, impacting upon cellular processes such as insulin secretion, gastric emptying, neurotransmission and epithelial repair. This review focuses on the molecular, structural and biological properties of GPR39 and its various physiological functions.


Dyspnoea and quality of life in older people at home.
To determine the prevalence of dyspnoea in older people at home, measure its impact on function and quality of life, and identify associated cardio-respiratory diseases. Cross-sectional population-based study. We sent a modified Medical Research Council (MRC) dyspnoea questionnaire to identify breathlessness in 1404 randomly selected subjects from general practitioner lists of 5002 subjects aged 70 years and over living at home. We visited a further random sample of 500 of these subjects at home and at a study centre. Community-based study in South Wales. Prevalence of dyspnoea (MRC grades 3-5) and its effect on psychological and functional status, and quality of life as measured by Hospital Anxiety and Depression, Nottingham Extended Activities of Daily Living and SF-36 questionnaires. The prevalence of dyspnoea as defined was 32.3% (95% confidence intervals: 30.3, 34.3). Breathless subjects had poorer functional status than non-breathless subjects. They also had poorer physical and mental health and were more likely to be anxious and depressed. The prevalence of left ventricular systolic dysfunction, reversible airways disease and obesity were all higher in those with dyspnoea. Dyspnoea is common in older people. Given its profound adverse effect on people's lives, dyspnoea is an important public health issue.


MRI technique for measurement of velocity vectors, acceleration, and autocorrelation functions in turbulent flow.
In this paper, we show velocity images for liquid flow in a cylindrical pipe at Reynolds numbers of up to 5000. Three orthogonal velocity components are acquired from a single excitation over a timescale of 60 ms thus obtaining an 'instantaneous' description of the fluid flow in steady laminar and chaotic turbulent flow. It is also shown how the technique, termed the gradient echo rapid velocity and acceleration imaging sequence (GERVAIS), can be used to acquire acceleration (convective derivative) images by incorporation of double pulsed gradient spin echo (PGSE) pairs into the pulse sequence such that the resulting phase shift is a measure of the change in the velocity over the mixing time between successive PGSE pairs. The technique has also been used to acquire up to 16 successive z-velocity images, providing spatio-temporal information of the velocity fluctuations over a period of 320 ms, which are then used to calculate spatially resolved velocity autocorrelation functions. It is found that over the timescale of the velocity measurements, which is principally determined by the residence time in the coil, the local velocity fluctuations are seen to be small compared to the local velocities, confirming that the data acquisition times achieved are rapid enough to capture 'instantaneous' images of the flow field.