[The evaluation of the role of endogenous Ca-dependent regulators and protein kinases in activating and inhibiting ion-transport ATPases].
The data on hormonal regulation of ATP-driving ion pumps are contradictory depending on the object used: whether native cells or isolated membranes. To eliminate this contrariety, we studied the ion transporting ATPases in saponin-permeabilized cells in the presence of all endogenous regulators. In permeabilized erythrocytes we obtained the presence of Ca(2+)-dependent activation of Ca(2+)-ATPase by factor(s) not affected by calmodulin antagonist R24571. We obtained also Ca(2+)-dependent activation and inhibition of Na+,K(+)-ATPase. At a concentration of Mg(2+)-ions corresponding to the intracellular level (370 microM), the 0.5-0.7 microM Ca(2+)-activated Na+,K(+)-ATPase (up to 3-fold), whereas the 1-5 microM Ca2+ inhibited it. The cyclic AMP (10(-5) M) inhibited or eliminated Ca(2+)-dependent activation. The decrease in Mg(2+)-ion concentration to 50 microM eliminated the activation and strengthened the inhibition, which reached 100% at the 1-2 microM Ca2+ concentration. The washing of membranes with EGTA eliminated Ca2+ effects on Na+,K(+)-ATPase. These data suggest that the ion-transporting ATPases are activated or inhibited by Ca(2+)-dependent regulators whose activities may be changed by protein kinase catalysed phosphorylation.


Convolution theory for dynamic systems: a bottom-up approach to the viscoelasticity of polymeric networks.
Biological materials such as the cytoskeleton are characterized by remarkable viscoelastic properties and therefore represent the subject of numerous micro- and macrorheological experimental studies. By generalizing the previously introduced dynamic convolution theory (DCT) to two dimensions, we devise a bottom-up approach for the viscoelastic properties of extended, crosslinked semiflexible polymer networks. Brownian dynamics (BD) simulations serve to determine the dynamic linear self- and cross-response properties of isolated semiflexible polymers to externally applied forces and torques; these response functions are used as input to the DCT. For a given network topology, the frequency-dependent response of the network subject to a given external force/torque distribution is calculated via the DCT allowing to resolve both micro- and macrorheological properties of the networks. A mapping on continuum viscoelastic theory yields the corresponding viscoelastic bulk moduli. Special attention is drawn to the flexibility of crosslinkers, which couple angular degrees of freedom at the network nodes and which are found to sensitively affect the resulting rheological properties of the polymeric meshwork.


Tether-directed bisfunctionalization reactions of c60 and c70.
Four easily isolable regio- and stereoselective bis-adducts of C60 and C70 , as well as a new C70 -dumbbell derivative, have been synthesized by using two different bismalonate tethered moieties. The derivatives that possess relatively long-tethered moieties show highly symmetric addition patterns, as evidenced by spectroscopic measurements, whereas the derivatives possessing the shorter-tethered moiety exhibit interesting addition patterns on C60 and C70 .


A role for the ventral hippocampal endocannabinoid system in fear-conditioned analgesia and fear responding in the presence of nociceptive tone in rats.
The endogenous cannabinoid (endocannabinoid) system plays an important role in fear-conditioned analgesia (FCA) and expression and extinction of conditioned fear. The hippocampus has an established role in both pain and conditioned fear and is a substrate for endocannabinoid activity. This study aimed to investigate the role of the endocannabinoid system in the ventral hippocampus (vHip) in FCA and in fear responding in the presence of nociceptive tone. Fear-conditioned rats displayed significantly increased freezing and 22-kHz ultrasonic vocalisation and a reduction in formalin-evoked nociceptive behaviour (ie, FCA) upon re-exposure to a context previously paired with footshock. Tissue levels of the endocannabinoids, anandamide, and 2-arachidonoylglycerol, as well as the fatty acid amide, palmitoylethanolamide, were significantly higher in the vHip of fear-conditioned rats compared with non-fear-conditioned controls. URB597 (inhibitor of fatty acid amide hydrolase [FAAH]), administered bilaterally into the vHip, significantly enhanced FCA during the entire trial and increased fear responding in formalin-treated rats early in the trial. The URB597-induced enhancement of FCA was blocked by intra-vHip administration of the cannabinoid(1) (CB(1)) receptor antagonist/inverse agonist rimonabant. Intra-vHip rimonabant alone had no effect on the expression of FCA, and URB597 did not significantly alter formalin-evoked nociceptive behaviour in non-fear-conditioned rats. These data suggest an important role for the endocannabinoid system in the vHip in FCA, whereby levels of 2-arachidonoylglycerol and the FAAH substrates palmitoylethanolamide and anandamide are increased in rats expressing FCA, and pharmacological inhibition of FAAH in the vHip enhances this form of endogenous analgesia via a CB(1) receptor-dependent mechanism.


Effects of extracellular signal-regulated kinase (ERK) on focal cerebral ischemia.
To determine the role of extracellular signal-regulated kinase (ERK)1/2 during focal cerebral ischemia. Left middle cerebral artery occlusion (MCAO) was undergone after the introduction of a nylon suture to the left internal carotid artery in 70 male adult CD-1 mice. ERK 1/2 phosphorylation was detected using Western blot analysis, and the morphological feature was determined by immunohistochemistry. An ERK pathway inhibitor, 1,4-diamino-2,3-dicyano-1,4-bis[2-amino-phenylthio] butadiene (U0126), was administered intravenously 20 minutes before MCAO, and the neurological deficit levels and the infarct volumes were measured 24 hours after MCAO. Phosphorylated ERK 1/2 (pERK 1/2) activity increased after 30 minutes of MCAO and peaked at 2 hours. The immunohistochemical study displayed a large number of pERK 1/2 positive cells in the ischemic basal ganglion and surrounding cortex. Double-labeled fluorescent staining identified the pERK1/2 positive cells as neurons or astrocytes. In U0126 treated mice which had undergone 24 hours of MCAO, the neurological deficit levels and the infarct volumes were 44.6% and 45.8% respectively, less than those of the control mice. ERK plays an important role in focal cerebral ischemia and inhibition of the ERK pathway can help protect against ischemic brain injury, which may provide a therapeutic approach for cerebral ischemia.


Oxidative stress implications for therapeutic vaccine development against Chagas disease.
Pathogenesis of Chagas disease (CD) caused by the protozoan parasite Trypanosoma cruzi (T. cruzi) involves chronic oxidative and inflammatory stress. In this review, we discuss the research efforts in therapeutic vaccine development to date and the potential challenges imposed by oxidative stress in achieving an efficient therapeutic vaccine against CD. This review covers the immune and nonimmune mechanisms of reactive oxygen species production and immune response patterns during T. cruzi infection in CD. A discussion on immunotherapy development efforts, the efficacy of antigen-based immune therapies against T. cruzi, and the role of antioxidants as adjuvants is discussed to provide promising insights to developing future treatment strategies against CD. Administration of therapeutic vaccines can be a good option to confront persistent parasitemia in CD by achieving a rapid, short-lived stimulation of type 1 cell-mediated immunity. At the same time, adjunct therapies could play a critical role in the preservation of mitochondrial metabolism and cardiac muscle contractility in CD. We propose combined therapy with antigen-based vaccine and small molecules to control the pathological oxidative insult would be effective in the conservation of cardiac structure and function in CD.


Cervicogenic headache: pathophysiology, diagnostic criteria and treatment.
Cervicogenic headache is a relatively common and still controversial form of headache arising from structures in the neck. Cervicogenic headache is a unilateral fixed headache characterised by pain that starts in the neck and spreads to the ipsilateral oculo-fronto-temporal area. The pathophysiology of cervicogenic headache probably depends on the effects of various local pain-producing or eliciting factors, such as intervertebral dysfunction, cytokines and nitric oxide. A reliable diagnosis of cervicogenic headache can be made based on the criteria established in 1998 by the Cervicogenic Headache International Study Group or the International Headache Society's most recent International Classification of Headache Disorders (2004). Various therapies have been used in the management of cervicogenic headache. These range from lowly invasive, drug-based therapies to highly invasive, surgical-based therapies. Unfortunately, the paucity of experimental models for cervicogenic headache and the relative lack of biomolecular markers for the condition mean much is still unclear about cervicogenic headache and the disorder remains inadequately treated.


Infrared Quantum Dots: Progress, Challenges, and Opportunities.
Infrared technologies provide tremendous value to our modern-day society. The need for easy-to-fabricate, solution-processable, tunable infrared active optoelectronic materials has driven the development of infrared colloidal quantum dots, whose band gaps can readily be tuned by dimensional constraints due to the quantum confinement effect. In this Perspective, we summarize recent progress in the development of infrared quantum dots both as infrared light emitters ( e.g., in light-emitting diodes, biological imaging, etc.) as well as infrared absorbers ( e.g., in photovoltaics, solar fuels, photon up-conversion, etc.), focusing on how fundamental breakthroughs in synthesis, surface chemistry, and characterization techniques are facilitating the implementation of these nanostructures into exploratory device architectures as well as in emerging applications. We discuss the ongoing challenges and opportunities associated with infrared colloidal quantum dots.


[Effect of specific inhibition of DNA replication by aphidicolin on primary differentiation of preimplantation mouse embryos].
Aphidicolin inhibits cleavage at every stage of preimplantation of Mouse embryos without affecting compaction. Blastulation is initiated only if the treatment is applied at the late 8-cell stage. It is concluded that the S phase of the 4th cell cycle might be critical for the commitment of the embryo for blastocyst formation. The 4th cycle thus corresponds to a "quantal cell cycle". Moreover, aphidicolin induces premature cavitation which occurs even when the embryos have not yet initiated cytokinesis.


In vitro kinetic studies of formation of antigenic advanced glycation end products (AGEs). Novel inhibition of post-Amadori glycation pathways.
Nonenzymatic protein glycation (Maillard reaction) leads to heterogeneous, toxic, and antigenic advanced glycation end products ("AGEs") and reactive precursors that have been implicated in the pathogenesis of diabetes, Alzheimer's disease, and normal aging. In vitro inhibition studies of AGE formation in the presence of high sugar concentrations are difficult to interpret, since AGE-forming intermediates may oxidatively arise from free sugar or from Schiff base condensation products with protein amino groups, rather than from just their classical Amadori rearrangement products. We recently succeeded in isolating an Amadori intermediate in the reaction of ribonuclease A (RNase) with ribose (Khalifah, R. G., Todd, P., Booth, A. A., Yang, S. X., Mott, J. D., and Hudson, B. G. (1996) Biochemistry 35, 4645-4654) for rapid studies of post-Amadori AGE formation in absence of free sugar or reversibly formed Schiff base precursors to Amadori products. This provides a new strategy for a better understanding of the mechanism of AGE inhibition by established inhibitors, such as aminoguanidine, and for searching for novel inhibitors specifically acting on post-Amadori pathways of AGE formation. Aminoguanidine shows little inhibition of post-Amadori AGE formation in RNase and bovine serum albumin, in contrast to its apparently effective inhibition of initial (although not late) stages of glycation in the presence of high concentrations of sugar. Of several derivatives of vitamins B1 and B6 recently studied for possible AGE inhibition in the presence of glucose (Booth, A. A., Khalifah, R. G., and Hudson, B. G. (1996) Biochem. Biophys. Res. Commun. 220, 113-119), pyridoxamine and, to a lesser extent, thiamine pyrophosphate proved to be novel and effective post-Amadori inhibitors that decrease the final levels of AGEs formed. Our mechanism-based approach to the study of AGE inhibition appears promising for the design and discovery of novel post-Amadori AGE inhibitors of therapeutic potential that may complement others, such as aminoguanidine, known to either prevent initial sugar attachment or to scavenge highly reactive dicarbonyl intermediates.


Management of secondary hyperparathyroidism in stages 3 and 4 chronic kidney disease.
To review approved treatment options for secondary hyperparathyroidism (SHPT) in patients with stages 3 and 4 chronic kidney disease (CKD). Recently published data on the diagnosis and treatment of SHPT in patients with CKD were critically assessed. Early detection of SHPT is critical for effective treatment. Approximately 40% of patients with stage 3 CKD and 80% of patients with stage 4 have SHPT due to low serum 1,25-dihydroxyvitamin D levels. Appropriate treatment involves suppression of parathyroid hormone (PTH) to normal levels with active vitamin D therapy and phosphate binders. Ergocalciferol or cholecalciferol should be used to correct 25-hydroxyvitamin D levels either before or during active vitamin D therapy. Active vitamin D analogues include calcitriol, doxercalciferol, and paricalcitol. Calcitriol is effective, but has a narrow therapeutic window at higher doses because of hypercalcemia and hyperphosphatemia, which require frequent monitoring. Doxercalciferol is also effective, but has been associated with significant elevations in serum phosphorus requiring greater use of oral phosphate binders. Paricalcitol effectively suppresses PTH with minimal impact on serum calcium and phosphorus. Limited data exist on the use of cinacalcet in treating SHPT in stages 3 and 4 CKD, and it is only approved for use in patients receiving dialysis. SHPT is an early and major complication of CKD. Treatment involves suppression of PTH to prevent metabolic bone disease, bone loss, and metabolic complications that may result in marked morbidity and mortality. Early detection of elevated PTH levels with appropriate intervention using active vitamin D therapy, even in the absence of elevated serum phosphorus and reduced serum calcium, is critical.


Transcriptional analysis of the candidate spermatogenesis gene Ube1y and of the closely related Ube1x shows that they are coexpressed in spermatogonia and spermatids but are repressed in pachytene spermatocytes.
Ube1y is a Y-linked gene transcribed in the testis, which maps to a region of the mouse Y required for normal spermatogonial proliferation. Ube1y, together with a ubiquitously expressed homologue on the X chromosome (Ube1x), encodes ubiquitin-activating enzyme E1, an enzyme essential for eukaryotic cell proliferation. Ube1y is thus a strong candidate for the Y function in spermatogonial proliferation. Using probes specific for the two genes, we have used Northern analysis and RNase protection to assess transcript levels throughout testis development and, by using germ cell-deficient XXSxr(a) testes and purified cell fractions, we have defined the testicular cell types in which transcription occurs. Ube1y transcripts are already detectable in the fetal testis at 12.5 dpc, with higher levels at 14.5 dpc and then falling to low levels by the time of birth. Postnatally levels rise sharply, peaking at 10 dpp. Analysis of XXSxr(a) testes indicates that the bulk of the Ube1y transcription is in germ cells. The analysis of purified cell fractions shows that X- and Y-encoded transcripts are present in A spermatogonia, both are at very low levels (or perhaps absent) in pachytene spermatocytes and then return to high levels in round spermatids. The reactivation of transcription in round spermatids implies a requirement for the ubiquitination pathway at this time. The presence of Ube1x transcripts in A spermatogonia raises the question as to why Ube1y transcripts are required. This question is discussed in relation to the spermatogenic failure in XSxr(b)O mice which are deleted for Ube1y and it is argued that Ube1y serves to increase UBE1 production at a time of high demand. Ube1y transcripts were also detected in XXY and XY ovaries.


Helicobacter pylori eradication prevents secondary gastric cancer in patients with mild-to-moderate atrophic gastritis.
Whether Helicobacter pylori eradication prevents metachronous recurrence after endoscopic resection (ER) of early gastric cancer remains controversial. This multicenter retrospective study aimed to evaluate the long-term (> 5 years) effects of H. pylori eradication by stratifying patients' baseline degrees of atrophic gastritis. A total of 483 H. pylori-positive patients who had undergone ER for early gastric cancer were divided into two groups-(i) those having undergone successful H. pylori eradication within 1 year after ER (eradicated group, n = 294) and (ii) those with failed or not attempted H. pylori eradication (non-eradicated group, n = 189). The cumulative incidences of metachronous gastric cancer between the two groups were compared for all patients, for patients with mild-to-moderate atrophic gastritis (n = 182), and for patients with severe atrophic gastritis (n = 301). During a median follow-up of 5.2 years (range 1.1-14.8), metachronous cancer developed in 52 (17.7%) patients in the eradicated group and in 35 (18.5%) patients in the non-eradicated group (P = 0.11, log-rank test). In patients with mild-to-moderate atrophic gastritis (111 and 71 in the eradicated and non-eradicated groups, respectively), the cumulative incidence of metachronous cancer was significantly lower in the eradicated group than that in the non-eradicated group (P = 0.03, log-rank test). However, no significant intergroup difference was observed in patients with severe atrophic gastritis (P = 0.69, log-rank test). Helicobacter pylori eradication had a preventive effect on the development of metachronous gastric cancer in patients with mild-to-moderate atrophic gastritis.


Cell lineage-specific expression and function of the empty spiracles gene in adult brain development of Drosophila melanogaster.
The empty spiracles (ems) gene, encoding a homeodomain transcription factor, is a member of the cephalic gap gene family that acts in early specification of the anterior neuroectoderm in the embryonic brain of Drosophila. Here we show that ems is also expressed in the mature adult brain in the lineage-restricted clonal progeny of a single neuroblast in each brain hemisphere. These ems-expressing neuronal cells are located ventral to the antennal lobes and project a fascicle to the superior medial protocerebrum. All adult-specific secondary neurons in this lineage persistently express ems during postembryonic larval development and continue to do so throughout metamorphosis and into the adult. Mosaic-based MARCM mutant analysis and genetic rescue experiments demonstrate that ems function is autonomously required for the correct number of cells in the persistently expressing adult-specific lineage. Moreover, they indicate that ems is also required cell autonomously for the formation of the correct projections in this specific lineage. This analysis of ems expression and function reveals novel and unexpected roles of a cephalic gap gene in translating lineage information into cell number control and projection specificity in an individual clonal unit of the adult brain.


Calcification of the vas deferens and seminal vesicles: a review.
Calcification of the vas deferens and seminal vesicles is a rare condition of unknown incidence. It has been described in association with diabetes, hyperparathyroidism and genitourinary tuberculosis, amongst other conditions. Little is known about the clinical significance and management of this condition. We review the literature in an effort to find answers about an entity that is frequently appreciated as an incidental finding. An electronic database search was performed (Medline) using the key words: vas deferens; seminal vesicles; calcification, alone or in combination. Articles were selected according to relevance and quality of evidence. The search included published manuscripts between 1960 and 2012. A total of 17 relevant publications were identified. The majority were written in the English language and mostly consisted of case presentations and reports of radiologic findings. Calcification of the vas deferens and seminal vesicles is a rare condition. However, it may be implicated in male factor infertility and symptoms from the urogenital tract. Treatment should be directed towards the underlying cause on an individual basis. It is unknown whether control of the primary process has any effects on the histopathological appearance of the ducts and/or their improvement of function.


Tracheal epithelial cell-exosome-derived MiR-21-5p inhibits alveolar macrophage pyroptosis to resist pulmonary bacterial infection through PIK3CD-autophagy pathway.
Structural cells play an important role in regulating immune cells during infection. Our aim was to determine whether structural porcine tracheal epithelial cells (PTECs) can regulate alveolar macrophages (AMs) to prevent bacterial pneumonia, explore the underlying mechanism(s) and therapeutic target. Actinobacillus pleuropneumoniae (APP) was used as the model strain for infection studies. Small RNA sequencing was used to identify differentially abundant exosome-derived miRNAs. The role of PTECs exosome-derived miR-21-5p in regulating AMs autophagy, pyroptosis, reactive oxygen species (ROS) was determined using RT-qPCR, western-blotting, flow cytometry, immunohistochemistry. Luciferase reporter assays were conducted to identify potential binding targets of miR-21-5p. The universality of miR-21-5p action on resistance to bacterial pulmonary infection was demonstrated using Klebsiella pneumoniae or Staphylococcus aureus in vitro and in vivo infection models. MiR-21-5p was enriched in PETCs-derived exosomes, which protected AMs against pulmonary bacterial infection. Mechanistically, miR-21-5p targeted PIK3CD, to promote autophagy of AMs, which reduced the pyroptosis induced by APP infection via inhibiting the over-production of ROS, which in turn suppressed the over-expression of pro-inflammatory cytokines, and increased bacterial clearance. Importantly, the protective effect and mechanism of miR-21-5p were universal as they also occurred upon challenge with Klebsiella pneumoniae and Staphylococcus aureus. Our data reveals miR-21-5p can promote pulmonary resistance to bacterial infection by inhibiting pyroptosis of alveolar macrophages through the PIK3CD-autophagy-ROS pathway, suggesting PIK3CD may be a potential therapeutic target for bacterial pneumonia.


Clinically prescribed orthoses demonstrate an increase in velocity of gait in children with cerebral palsy: a retrospective study.
The purpose of this study was to determine the effect clinically prescribed ankle-foot orthoses (AFOs) have on the temporal-spatial parameters of gait, as compared with barefoot walking in children with cerebral palsy. A retrospective chart review of data collected between 1995 and 1999 in our motion analysis laboratory was performed. A retrospective chart review of 700 patients revealed 115 patients (mean age 9 years) who had a primary diagnosis of CP (diplegia n=97, hemiplegia n=18). All were wearing clinically prescribed hinged or solid AFOs at the time of undergoing a three dimensional gait analysis. In line with our standard clinical practice, data for both conditions (braced and barefoot walking) were collected the same day by the same examiner. Statistical analyses indicated the temporal and spatial gait parameters of velocity, stride length, step length, and single limb stance were significantly increased (p<0.001) with the use of AFOs versus barefoot walking. Cadence was the only parameter found to not be statistically different.


Comparison of Meconium DNA Extraction Methods for Use in Microbiome Studies.
The establishment of human gut microbiota commences initially in utero. Meconium-the first fecal material passed after birth-can be used to study fetal gut contents; however, processing meconium samples for microbiome studies presents significant technical challenges. Meconium hosts a low biomass microbiome, is tar-like in texture and contains high concentrations of PCR inhibitors. This study aimed to evaluate four different DNA extraction methods to elucidate the most effective method for bacterial DNA recovery and sequencing analysis from first-pass meconium. Samples from five infants were collected and processed using the following extraction kits: (1) Qiagen QIAamp DNA Stool Mini (QS); (2) Qiagen QIAamp DNA Microbiome (QM); (3) MoBio PowerSoil (PS); (4) MoBio MagAttract PowerMicrobiome (PM). Additionally, Kit PM was employed with a double inhibitor removal treatment (IRT) step (PM2). Bacterial DNA recovery was assessed by qPCR. Any PCR inhibition in samples was measured by spiking DNA eluates with 0.1 ng of pure Streptococcus agalactiae (GBS) DNA followed by qPCR quantitation. Kit PM yielded the highest average total DNA yield (79.3 ng per gram of meconium). Samples extracted with kit PS had the highest detectable levels of 16S rRNA gene by qPCR. The ability of each kit to overcome PCR inhibition varied, with qPCR on GBS-spiked DNA from kits QS, QM, PS, and PM recovering 87.1, 91.0, 88.8, and 37.9% GBS DNA, respectively. Double IRT improved the performance of kit PM, increasing GBS recovery to 56.5%. However, once DNA yield was normalized to the level recovered with the other kits 100% of GBS DNA was detected, suggesting that levels of PCR inhibitors are related to DNA yield from kit PM. Ion Torrent 16S rRNA gene sequencing revealed a high level of inter-kit variation in meconium microbiome structure. In particular, kit QM showed a bias toward extracting Firmicute DNA, while the other kits extracted primarily Proteobacterial DNA. Choice of extraction kit greatly impacts on the ability to extract and detect bacterial DNA in meconium and on the microbiome community structure generated from these samples.


Intact specimen extraction in laparoscopic nephrectomy procedures: Pfannenstiel versus expanded port site incisions.
Laparoscopic nephrectomy is considered the standard of care for most Stage T1 and T2 renal tumors. Most centers perform intact extraction rather than morcellation. The extraction incision location varies, with no consensus on the best site. We compared the operative and perioperative parameters after transperitoneal laparoscopic nephrectomy procedures with intact specimen extraction through a Pfannenstiel (PFN) or expanded port site (EPS) incision. The consecutive charts of 150 patients (March 2001 to October 2003) undergoing laparoscopic radical nephrectomy (LRN), laparoscopic nephroureterectomy, or laparoscopic donor nephrectomy with intact specimen extraction were reviewed. The specimens were extracted by way of a PFN or an EPS incision. Two analyses were completed. The first included only LRN, and the second included LRN, laparoscopic nephroureterectomy, and laparoscopic donor nephrectomy. In the LRN-only analysis, the PFN group had a shorter hospital stay (2.84 versus 3.37 days, P <0.05). This group also used significantly less morphine (23.7 versus 47.3 mg, P <0.006). The PFN group in the second analysis also used less morphine (26.3 versus 51.1 mg, P <0.002). Four extraction site complications were found; 1 patient in the PFN group developed cellulitis, and 3 patients in the EPS group developed an incisional hernia. This evidence suggests reduced morbidity with intact specimen extraction through a PFN incision compared with an EPS incision during laparoscopic nephrectomy procedures. Our practice has been modified on the basis of these data, and all specimens are now removed through a PFN incision when suitable. Urologists should consider PFN incisions for specimen extraction with laparoscopic nephrectomy procedures.


Detection of Blastocystis in clinical stool specimens using three different methods and morphological examination in Jones' medium.
Blastocystis was identified almost a century ago, yet its biology and pathogenicity status in humans is obscure. Studies on Blastocystis in India are scanty and are mostly microscopy based. This study compared three detection modalities to determine their efficiency in the identification of Blastocystis in human feces. A total of 279 stool samples were screened using microscopy, culture (Jones' medium), and polymerase chain reaction (PCR)-based methods. Among the three, PCR is considered the gold standard test for detection of Blastocystis, as it helps to authenticate the sensitivity, specificity, and kappa agreement obtained by the other two tests. The morphological features of Blastocystis were recorded at 24, 48, and 72 h. After positive morphological identification, ten samples were cultured on Löwenstein-Jensen (LJ) medium and Locke's egg slant medium. The sensitivity and specificity determined on the basis of microscopy were 36.2% and 99.4%, respectively. On the other hand, Jones' medium showed 67.6% sensitivity and 100% specificity. Further, we documented various morphological and reproductive features of Blastocystis using various staining techniques on cultures positive in Jones' medium. In addition, we also found that LJ medium was not equally efficacious as Jones' medium in assisting the growth of Blastocystis. Although molecular diagnosis is a necessary tool for understanding the true epidemiology of Blastocystis, in laboratories devoid of molecular detection facilities, stool microscopy in conjunction with stool culture on Jones' medium could serve as the best alternative tool for the detection of Blastocystis.


Saralasin acetate test in renal transplant hypertension. Report of 17 cases and a review of the literature.
The saralasin acetate test was performed in 17 hypertensive patients with renal transplants. These results were compared with 39 previously published reports of transplant patients who had been tested in the same manner. Eighty-two percent of our patients had a positive saralasin acetate test, suggesting renin-dependent hypertension. Baseline plasma renin activity (PRA) was significantly higher in patients with positive tests (6.96 +/- 1.75 v 2.88 +/- 0.53 ng/mL/hr). However, positive tests were obtained in several patients who had normoreninemia, and PRA levels did not correlate with the magnitude of vasodepressor BP response to saralasin. Transplant artery stenosis, acute rejection, and chronic rejection were the most common posttransplant complications associated with a positive test, but several patients had hypertension alone. While highly sensitive for renin-dependent hypertension posttransplantation, the test had poor specificity for identification of any one cause of posttransplant hypertension.


One-year treatment continuation in patients prescribed risperidone long-acting injection in New Zealand: a retrospective study.
Treatment discontinuation is a major problem in routine clinical settings and is associated with poorer outcomes for conditions like schizophrenia. Risperidone long-acting injection (RLAI) has shown low discontinuation and good tolerability in long-term clinical trials. This retrospective study investigated RLAI continuation in a naturalistic clinical setting in New Zealand. Those starting publicly funded RLAI between October 1, 2005 and October 31, 2006 in five public mental health services were included in the study. Data were retrospectively collected for 443 patients 12 months pre- and post-RLAI initiation. Patient demographics, diagnosis, antipsychotic treatment and treatment setting were obtained from clinical files and patient information systems. Patients were mostly male (64%), the mean age was 35.9 years, and were predominantly European (43%) or Māori (29%). Most started RLAI due to adherence issues (77%). Adverse events due to RLAI were reported by 39%; most commonly, extrapyramidal side effects. Fifty-eight percent of patients continued treatment 12 months after starting; patient choice (either refusal or failure to turn up) was the most common reason for discontinuation (54%), followed by lack of efficacy (29%). Alternative antipsychotic treatment was prescribed for 79% of those who discontinued. Regression analysis found continuation was more common for people started in the community, on a compulsory treatment order, with a dose of >25 mg and for non-Māori. RLAI appears well-tolerated and over half the patients continued treatment for one year or more. Using treatment continuation as an indicator of effectiveness, RLAI appears to be an effective treatment for people with adherence problems in this real world practice setting.


Autologous nonmyeloablative hematopoietic stem cell transplantation for neuromyelitis optica.
To determine if autologous nonmyeloablative hematopoietic stem cell transplantation (HSCT) could be a salvage therapy for neuromyelitis optica spectrum disorder (NMOSD). Thirteen patients were enrolled in a prospective open-label cohort study (11 NMOSD aquaporin-4-immunoglobulin G [AQP4-IgG]-positive, 1 NMOSD without AQP4, and 1 NMOSD AQP4-IgG-positive with neuropsychiatric systemic lupus erythematosus [SLE]). Following stem cell mobilization with cyclophosphamide (2 g/m2) and filgrastim, patients were treated with cyclophosphamide (200 mg/kg) divided as 50 mg/kg IV on day -5 to day -2, rATG (thymoglobulin) given IV at 0.5 mg/kg on day -5, 1 mg/kg on day -4, and 1.5 mg/kg on days -3, -2, and -1 (total dose 6 mg/kg), and rituximab 500 mg IV on days -6 and +1. Unselected peripheral blood stem cells were infused on day 0. AQP4-IgG antibody status was determined by Clinical Laboratory Improvement Amendments-validated ELISA or flow cytometry assays. Cell-killing activity was measured using a flow cytometry-based complement assay. Median follow-up was 57 months. The patient with coexistent SLE died of complications of active lupus 10 months after HSCT. For the 12 patients with NMOSD without other active coexisting autoimmune diseases, 11 patients are more than 5 years post-transplant, and 80% are relapse-free off all immunosuppression (p < 0.001). At 1 and 5 years after HSCT, Expanded Disability Status Scale score improved from a baseline mean of 4.4 to 3.3 (p < 0.01) at 5 years. The Neurologic Rating Scale score improved after HSCT from a baseline mean of 69.5 to 85.7 at 5 years (p < 0.01). The Short Form-36 health survey for quality of life total score improved from mean 34.2 to 62.1 (p = 0.001) at 5 years. In the 11 patients whose baseline AQP4-IgG serostatus was positive, 9 patients became seronegative by the immunofluorescence or cell-binding assays available at the time; complement activating and cell-killing ability of patient serum was switched off in 6 of 7 patients with before and after HSCT testing. Two patients remained AQP4-IgG-seropositive (with persistent complement activating and cell-killing ability) and relapsed within 2 years of HSCT. No patient with seronegative conversion relapsed. Prolonged drug-free remission with AQP4-IgG seroconversion to negative following nonmyeloablative autologous HSCT warrants further investigation.


PKC activation is required by EGF-stimulated Na(+)-H+ exchanger in human pleural mesothelial cells.
Epidermal growth factor (EGF) stimulates the Na(+)-H+ exchanger, leading to enhanced cell proliferation. In human pleural mesothelial cells (PMCs), the intracellular signaling mechanism mediating the EGF-induced stimulation of the Na(+)-H+ exchanger has not yet been identified. Using a pH-sensitive fluorescent probe, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, to measure changes in intracellular pH (pHi), we found that 1) EGF and 12-O-tetradecanoylphorbol 13-acetate (TPA; a phorbol ester) both stimulate the ethylisopropyl amiloride-sensitive Na(+)-H+ exchanger; 2) TPA-induced alkalosis can be blocked by protein kinase C (PKC) inhibitors (chelerythrine and staurosporine) or by PKC down-regulation, indicating that PKC activation is involved in the stimulation of the Na(+)-H+ exchanger. However, TPA-induced alkalosis is not blocked by tyrosine kinase inhibitors; and 3) the stimulatory effect of EGF on the Na(+)-H+ exchanger acts via stimulation of tyrosine kinase-receptor activity because it is inhibited by tyrosine kinase inhibitors (genistein, lavendustin A, and herbimycin A). It also involves PKC activation because EGF-induced alkalosis was blocked by PKC inhibitors. These results suggest that PKC activation is one of the downstream signals for EGF-induced activation of the Na(+)-H+ exchanger in primary cultures of human pleural mesothelial cells.


Evaluation of Smoky Taste in Cocoa Powder.
The aim of this study was to determine the levels of nine phenols (phenol, guaiacol derivatives, xylenols, and cresols) by using a GC/MS technique in smoked samples under control, commercial samples taken from contaminated batches, and uncontaminated samples. The smoky taste of cocoa powder may have two origins: drying and storage. The sample was steam distilled, extracted with ethyl ether, concentrated, and chromatographed on a 50-m OV-351 fused silica column. The physicochemical data obtained were related to sensory evaluation and submitted to multivariate distribution (Mahalanobis distance) and Pearson Chi-squared test. The discriminatory phenols identified were phenol, 3-methylphenol (m-cresol), 2,3-dimethylphenol (2,3-xylenol), 3-ethylphenol, 4-ethylphenol, and total phenols. The results obtained imply that uncontaminated smoked cocoa samples should have the following maximum concentrations: phenol, 2 mg/kg; 3-methylphenol, 0.9 mg/kg; 2,3-dimethylphenol, 0.55 mg/kg; 3-ethylphenol, 0.90 mg/kg; 4-ethylphenol, 0.70 mg/kg; and total phenols, 9.6 mg/kg.


Development and validation of a risk model for prediction of hazardous alcohol consumption in general practice attendees: the predictAL study.
Little is known about the risk of progression to hazardous alcohol use in people currently drinking at safe limits. We aimed to develop a prediction model (predictAL) for the development of hazardous drinking in safe drinkers. A prospective cohort study of adult general practice attendees in six European countries and Chile followed up over 6 months. We recruited 10,045 attendees between April 2003 to February 2005. 6193 European and 2462 Chilean attendees recorded AUDIT scores below 8 in men and 5 in women at recruitment and were used in modelling risk. 38 risk factors were measured to construct a risk model for the development of hazardous drinking using stepwise logistic regression. The model was corrected for over fitting and tested in an external population. The main outcome was hazardous drinking defined by an AUDIT score ≥8 in men and ≥5 in women. 69.0% of attendees were recruited, of whom 89.5% participated again after six months. The risk factors in the final predictAL model were sex, age, country, baseline AUDIT score, panic syndrome and lifetime alcohol problem. The predictAL model's average c-index across all six European countries was 0.839 (95% CI 0.805, 0.873). The Hedge's g effect size for the difference in log odds of predicted probability between safe drinkers in Europe who subsequently developed hazardous alcohol use and those who did not was 1.38 (95% CI 1.25, 1.51). External validation of the algorithm in Chilean safe drinkers resulted in a c-index of 0.781 (95% CI 0.717, 0.846) and Hedge's g of 0.68 (95% CI 0.57, 0.78). The predictAL risk model for development of hazardous consumption in safe drinkers compares favourably with risk algorithms for disorders in other medical settings and can be a useful first step in prevention of alcohol misuse.


Antihypertensive therapy and cardiovascular complications.
Hypertension is a quantitative derangement of a physiological parameter rather than a disease and acceptance of this fact has greatly influenced the treatment of hypertension. Experience gathered over many years can be critically analysed to gain further information about the treatment of hypertension and the effect of different treatments on the complications of hypertension. The extent to which hypertension is a causative risk factor for coronary heart disease is still disputed, a lowering of blood pressure having not yet been conclusively shown to be invariably associated with a reduction in the incidence of coronary heart disease. On the other hand, a causative relationship with cerebro-vascular disease has been convincingly shown. This raises the possibility that, at least for coronary heart disease, the way in which blood pressure is lowered may be important. Many treatments for hypertension affect other parameters, e.g., electrolytes and plasma lipids, and it may be that a positive effect on blood pressure may be offset by a negative effect on another parameter. Evidence that this is indeed the case with diuretics has emerged from a number of large-scale trials. The cardio-protective effect of beta blockers has also recently been extensively examined, although no clear conclusions have yet been reached. Primary prevention and secondary prevention are not the same, and positive results in one do not necessarily mean that the same drug will be effective in the other. A number of trials have been carried out to gain more information about the value of beta blockers, but it is unlikely that conclusive evidence will emerge due to the small difference in the therapies compared and the relatively short duration of the trials.


Purine receptor antagonist modulates serology and affective behaviors in lupus-prone mice: evidence of autoimmune-induced pain?
Neurologic and psychiatric (NP) manifestations are severe complications of systemic lupus erythematosus (SLE). As commonly seen in patients, spontaneous disease onset in the MRL/MpJ-Fas(lpr)/J (MRL-lpr) mouse model of NP-SLE is accompanied by increased autoantibodies, pro-inflammatory cytokines and behavioral dysfunction which precede neuroinflammation and structural brain lesions. The role of purinergic receptors in the regulation of immunity and behavior remains largely unexplored in the field of neuropsychiatry. To examine the possibility that purinoception is involved in the development of affective behaviors, the P2X purinoceptor antagonist, suramin, was administered to lupus-prone mice from 5 to 14 weeks of age. In addition to food and water measures, novel object and sucrose preference tests were performed to assess neophobic anxiety- and anhedonic-like behaviors. Enzyme-linked immunosorbant assays for anti-nuclear antibodies (ANA) and pro-inflammatory cytokines were employed in immunopathological analyses. Changes in dendritic morphology in the hippocampal CA1 region were examined by a Golgi impregnation method. Suramin significantly lowered serum ANA and prevented behavioral deficits, but did not prevent neuronal atrophy in MRL-lpr animals. In a new batch of asymptomatic mice, systemic administration of corticosterone was found to induce aberrations in CA1 dendrites, comparable to the "stress" of chronic disease. The precise mechanism(s) through which purine receptor inhibition exerted beneficial effects is not known. The present data supports the hypothesis that activation of the peripheral immune system induces nociceptive-related behavioral symptomatology which is attenuated by the analgesic effects of suramin. Hypercortisolemia may also initiate neuronal damage, and metabolic perturbations may underlie neuro-immuno-endocrine imbalances in MRL-lpr mice.


Protocol for precision editing of endogenous Chlamydomonas reinhardtii genes with CRISPR-Cas.
CRISPR-Cas genome engineering in the unicellular green algal model Chlamydomonas reinhardtii has until recently suffered from low integration efficiencies despite traditional genetics being well established. Here, we present a protocol for efficient homology-directed knockin mutagenesis in all commonly used strains of Chlamydomonas. We describe steps for scarless integration of fusion tags and sequence modifications of almost all proteins without the need for a preceding mutant line. We further empower this genetic-editing approach by efficient crossing and highly robust screening protocols. For complete details on the use and execution of this protocol, please refer to Nievergelt et al. (2023).1.


Innate immune activation in the pathogenesis of a murine model of globoid cell leukodystrophy.
Globoid cell leukodystrophy is a lysosomal storage disease characterized by the loss of galactocerebrosidase. Galactocerebrosidase loss leads to the accumulation of psychosine and subsequent oligodendrocyte cell death, demyelination, macrophage recruitment, and astroglial activation and proliferation. To date, no studies have elucidated the mechanism of glial cell activation and cytokine and chemokine up-regulation and release. We explored a novel explanation for the development of the pathological changes in the early stages of globoid cell leukodystrophy associated with toll-like receptor (TLR) 2 up-regulation in the hindbrain and cerebellum as a response to dying oligodendrocytes. TLR2 up-regulation on microglia/macrophages coincided with morphological changes consistent with activation at 2 and 3 weeks of age. TLR2 up-regulation on activated microglia/macrophages resulted in astrocyte activation and marked up-regulation of cytokines/chemokines. Because oligodendrocyte cell death is an important feature of globoid cell leukodystrophy, we tested the ability of TLR2 reporter cells to respond to oligodendrocyte cell death. These reporter cells responded in vitro to medium conditioned by psychosine-treated oligodendrocytes, indicating the likelihood that oligodendrocytes release a TLR2 ligand during apoptosis. TLRs are a member of the innate immune system and initiate immune and inflammatory events; therefore, the identification of TLR2 as a potential driver in the activation of central nervous system glial activity in globoid cell leukodystrophy may provide important insight into its pathogenesis.


Commercial insurance delays direct-acting antiviral treatment for hepatitis C kidney transplantation into uninfected recipients.
The advent of direct-acting antivirals (DAAs) has created an avenue for transplantation of hepatitis C virus (HCV)-infected donors into uninfected recipients (D+/R-). The donor transmission of HCV is then countered by DAA administration during the post-operative period. However, initiation of DAA treatment is ultimately dictated by insurance companies. A retrospective chart review of 52 D+/R- kidney recipients who underwent DAA treatment post-transplant was performed. Patients were grouped according to their prescription coverage plans, managed by either commercial or government pharmacy benefit managers (PBMs). Thirty-nine patients had government PBMs and 13 had commercial PBMs. Demographics were similar between the two groups. All patients developed HCV viremia, but cleared the virus after treatment with DAA. Patients with government PBMs were treated earlier compared to those with commercial PBMs (11 days vs 26 days, P = .01). Longer time to DAA initiation resulted in higher peak viral loads (β = 0.39, R2 = .15, P = .01) and longer time to HCV viral load clearance (β = 0.41, R2 = .17, P = .01). D+/R- transplantation offers patients an alternative strategy to increase access. However, treatment can be profoundly delayed by a third-party payer authorization process that may be subjecting patients to unnecessary risks and worsened outcomes.


Biweekly prophylactic doxycycline for travelers' diarrhea.
A double-blind study to determine the efficacy of biweekly oral doxycycline in the prevention of travelers' diarrhea was conducted among 46 Peace Corps volunteers during their first six weeks in Honduras. The volunteers took either 100 mg of doxycycline per dose or a placebo for three weeks and were observed for an additional three weeks. There was no significant difference in the number of persons with travelers' diarrhea in the two groups (eight of 24 in the doxycycline group and 10 of 22 in the placebo group) in the three weeks when the drug was taken. However, significantly fewer episodes (P less than 0.05) of travelers' diarrhea occurred in the doxycycline group than in the placebo group at the end of the second, third, and fourth weeks. Enterotoxigenic Escherichia coli (ETEC) was the most common pathogen identified. ETEC from 13 (62%) of 21 stool samples were resistant to doxycycline. Biweekly doxycycline was only marginally effective in preventing travelers' diarrhea and did not prevent diarrhea secondary to doxycycline-resistant ETEC.


Thin aggregative fimbriae enhance Salmonella enteritidis biofilm formation.
Salmonella enteritidis enteropathogens produce a variety of potentially adherent fimbrial types including SEF14, SEF17, SEF18 and SEF21 (type I). In a simplified, pure culture, biofilm generating system the virulent isolate, S. enteritidis 3b, readily adhered to Teflon (polytetrafluoroethylene) and stainless steel forming thick cell aggregates. The inability of an isogenic SEF17-deficient mutant to form thick biofilms suggested a role for SEF17 in stabilizing cell-cell interactions during biofilm formation. Epifluorescent detection of SEF17 in biofilms confirmed the association of these fimbriae with aggregated cells but not with adherent mutants unable to produce SEF17. The reduced adherence observed with an isogenic SEF14/SEF21-deficient strain implicated the involvement of additional cell surface adherence factors, possibly including SEF21 (type I) fimbriae in the adherence of S. enteritidis to stainless steel or Teflon. The role of SEF17 fimbriae in biofilm formation and the contributions of SEF17 to the persistence of Salmonellae on surfaces and in food are discussed.


Modified ammonia electrode method to investigate D-asparagine breakdown by Campylobacter strains.
An ammonia electrode method has been developed for investigating the deamination of amino acids by bacteria. It consists of incubating a standard inoculum of organisms in an amino acid solution and then measuring the amount of ammonia evolved by the electrode. Two hundred and twelve Campylobacter strains (118 C. jejuni and 94 C. coli) were tested for their ability to break down D-asparagine by this method. Organism control (bacterial suspension in buffer alone) values ranged from 0.44 to 2.0 (mean 0.93 +/- 0.24) ammonia concentration (AC) units (one AC unit is equal to 10(-5) mol of ammonia per liter), whereas test values ranged from 0.60 to 46.0 units. Test ACs of less than 2 units (97 strains) were considered negative, whereas ACs of greater than or equal to 10 (77 strains) were considered positive for D-asparaginase; 38 (18%) strains with ACs between 2 and 10 units were provisionally assigned an intermediate status. The amount of ammonia produced by strains with ACs of greater than or equal to 10 increased greatly when the inoculum size was increased, whereas this was not a feature of strains with ACs of less than 2 units. The presence or absence of an inoculum effect was instrumental in classifying strains with intermediate ACs and allowed a breakpoint to be defined. When the ammonia electrode method was repeated, 97.6% of the 212 strains gave the same positive or negative reaction that they did on the first occasion. Thus the test was highly reproducible. Five strains (all porcine C. coli from Germany) were unclassifiable because they repeatedly gave either a weak-positive or negative reaction. Overall, 12.7% of C. jejuni strains and 86.2% of C. coli strains were positive for D-asparaginase. The ammonia electrode method was found to be simple and reliable for separating strains on the basis of D-asparaginase activity.


Health status and potential uptake of transgenic DNA by Japanese quail fed diets containing genetically modified plant ingredients over 10 generations.
The hypothesis assumes that feed containing GMOs affects animal health and results in the transgene product accumulating in the body. Therefore, the objective of the study was to evaluate the impact of genetically modified (GM) ingredients used in poultry diets on aspects of bird health status and accumulation of transgenic DNA in eggs, breast muscle and internal organs. A total of 10 generations of Japanese quail were fed three types of diets: group A - containing GM soya (Roundup Ready) and non-GM maize, group B - containing GM maize (MON810) and non-GM soya, and group C - containing non-GM soya and maize. Bird performance traits were monitored throughout the trial. In 17-week-old animals of each generation, health examination took place on birds from each group including post-mortem necropsy and histological organ evaluation. For the purpose of transgenic DNA detection, samples of selected important tissues were taken. A molecular screening method of PCR amplification was used. The analysis of the sectional examination of birds used in the current experiment did not indicate the existence of the pathological changes caused by pathogens, nutritional factors or of environmental nature. The histopathological changes occurred in all three dietary groups and there were no statistically significant differences between the groups. There was no transgene amplification - neither CaMV35S promoter sequence nor nos terminator sequence, in the samples derived from breast muscle, selected tissues and germinal discs (eggs). According to the obtained results, it was concluded that there was no negative effect of the use of GM soya or maize with regard to bird health status or to the presence of transgenic DNA in the final consumable product.


Do Fair and Just Systems Require Compensation for the Disadvantages of the Natural Lottery? A Discussion on Society's Duties on the Provision of Gene Therapy.
Genetic diseases have been thought to be acquired as a result of sheer bad luck. However, recent advances in medical science have demonstrated the mechanisms of genetic disorders, which enable us to intervene with their occurrence and treatment. Today, gene therapy, once considered too risky, has become safer and can save the lives of patients with previously untreatable and lethal genetic diseases. However, the positive expectations from gene therapy are overshadowed by their extremely high prices. Thus, the duty of society in the provision of gene therapies has been frequently discussed. The discussions mainly focus on how to meet the genetic treatment needs of patients without violating the notion of justice and fairness in society. This study discusses the theoretical grounds for society's duty to compensate for genetic disease patients' disadvantages by providing them with appropriate genetic treatment. The main question is whether a fair and just system requires society to provide available lifesaving gene therapy to patients in need. The discussion is constructed on the crucial notion of the fair equal opportunity principle in a just system and the plausibility of including disadvantages emerging from bad luck in the natural lottery in the domain of justice.


Health effects of drought: a systematic review of the evidence.
Introduction. Climate change projections indicate that droughts will become more intense in the 21 century in some areas of the world. The El Niño Southern Oscillation is associated with drought in some countries, and forecasts can provide advance warning of the increased risk of adverse climate conditions. The most recent available data from EMDAT estimates that over 50 million people globally were affected by drought in 2011. Documentation of the health effects of drought is difficult, given the complexity in assigning a beginning/end and because effects tend to accumulate over time. Most health impacts are indirect because of its link to other mediating circumstances like loss of livelihoods. Methods. The following databases were searched: MEDLINE; CINAHL; Embase; PsychINFO, Cochrane Collection. Key references from extracted papers were hand-searched, and advice from experts was sought for further sources of literature. Inclusion criteria for papers summarised in tables include: explicit link made between drought as exposure and human health outcomes; all study designs/methods; all countries/contexts; any year of publication. Exclusion criteria include: drought meaning shortage unrelated to climate; papers not published in English; studies on dry/arid climates unless drought was noted as an abnormal climatological event. No formal quality evaluation was used on papers meeting inclusion criteria. Results. 87 papers meeting the inclusion criteria are summarised in tables. Additionally, 59 papers not strictly meeting the inclusion criteria are used as supporting text in relevant parts of the results section. Main categories of findings include: nutrition-related effects (including general malnutrition and mortality, micronutrient malnutrition, and anti-nutrient consumption); water-related disease (including E coli, cholera and algal bloom); airborne and dust-related disease (including silo gas exposure and coccidioidomycosis); vector borne disease (including malaria, dengue and West Nile Virus); mental health effects (including distress and other emotional consequences); and other health effects (including wildfire, effects of migration, and damage to infrastructure). Conclusions. The probability of drought-related health impacts varies widely and largely depends upon drought severity, baseline population vulnerability, existing health and sanitation infrastructure, and available resources with which to mitigate impacts as they occur. The socio-economic environment in which drought occurs influences the resilience of the affected population. Forecasting can be used to provide advance warning of the increased risk of adverse climate conditions and can support the disaster risk reduction process. Despite the complexities involved in documentation, research should continue and results should be shared widely in an effort to strengthen drought preparedness and response activities.


An Aminoacyl tRNA Synthetase, OKI1, Is Required for Proper Shoot Meristem Size in Arabidopsis.
In plants, the stem cells that form the shoot system reside within the shoot apical meristem (SAM), which is regulated by feedback signaling between the WUSCHEL (WUS) homeobox protein and CLAVATA (CLV) peptides and receptors. WUS-CLV feedback signaling can be modulated by various endogenous or exogenous factors, such as chromatin state, hormone signaling, reactive oxygen species (ROS) signaling and nutrition, leading to a dynamic control of SAM size corresponding to meristem activity. Despite these insights, however, the knowledge of genes that control SAM size is still limited, and in particular, the regulation by ROS signaling is only beginning to be comprehended. In this study, we report a new function in maintenance of SAM size, encoded by the OKINA KUKI1 (OKI1) gene. OKI1 is expressed in the SAM and encodes a mitochondrial aspartyl tRNA synthetase (AspRS). oki1 mutants display enlarged SAMs with abnormal expression of WUS and CLV3 and overaccumulation of ROS in the meristem. Our findings support the importance of normal AspRS function in the maintenance of the WUS-CLV3 feedback loop and SAM size.


Apolipoprotein E is a biologically active constituent of the normal immunoregulatory lipoprotein, LDL-In.
A normal plasma lipoprotein, termed LDL-In, has been shown to be a potent inhibitor of mitogen-driven human lymphocyte proliferation in vitro and of primary antibody responses in the mouse. To determine whether the immunoregulatory activity of LDL-In resided with the protein rather than the lipid constituents of LDL-In, one of the apoproteins of LDL-In, apoprotein E, was isolated from plasma and was analyzed for its inhibitory activity. Apoprotein E, isolated after delipidization of lipoproteins with either methyl ethyl ketone or ethanol and ethyl ether, was immunosuppressive. Furthermore, the characteristics of inhibition of cellular [3H]thymidine uptake by isolated apoprotein E were identical to those characteristics obtained with suppression by LDL-In. Inhibition by apoprotein E and LDL-In required preincubation of the cells with either apoprotein or lipoprotein for 24 hr before exposure of the cells to mitogen for maximal expression of suppressive activity, and this inhibition could not be reversed by removal of non-cell-associated inhibitor before stimulation. Neither apoprotein E or LDL-In was inhibitory when they were added to the cells after mitogen stimulation. The only difference noted between suppression by apoprotein E and LDL-In was that of dose. Compared with quantitative estimates of the apoprotein E content of LDL-In, significantly more isolated apoprotein E was required than lipoprotein-associated E for comparable levels of suppression. The potency of apoprotein E could be increased by adding it to cells in the presence of dimyristoylphosphatidylcholine/cholesterol vesicles. The data suggesting that phospholipid increased the specific activity of apoprotein E by altering its molecular dispersion was obtained from analyses of the interaction of apo E with cells, as well as the plastic culture vessels. The results suggested that the molecular dispersion and perhaps organization of isolated apoprotein E in an aqueous system is critical to its interaction with lymphocytes and subsequently its biological activity.


Familial autosomal recessive renal tubular acidosis: importance of early diagnosis.
Untreated renal tubular acidosis (RTA) can result in severe complications. We reviewed the clinical features of patients with mutations in two genes causing RTA and evaluated their developmental expression assuming that timing, symptom severity and complications may be related to its occurrence. Clinical data from 16 patients with RTA due to mutations in either ATP6V1B1 or CAII were retrospectively reviewed. Both genes' localization and expression pattern in the developing human kidney were analyzed by real-time polymerase chain reaction and immunostaining. RTA-presenting symptoms were non-specific. Patients with mutations in ATP6V1B1 had earlier presentation (4.9 vs. 11 months, p < 0.041) and longer time to diagnosis than patients with CAII mutations (5.8 vs. 57 months, p < 0.01). Patients with ATP6V1B1 mutations were more likely to develop chronic kidney disease than those with CAII mutations (follow-up GFR values: 89 vs. 110 ml/min/1.73 m2, respectively, p < 0.017), probably secondary to nephrocalcinosis. Both ATP6V1B1 and CAII were expressed early during human nephrogenesis, with relatively higher transcript levels of ATP6V1B1. There is considerable delay in establishing a diagnosis of both types of RTA, supporting the need for earlier biochemical investigation. RTA due to ATP6V1B1 mutations is associated with mild progressive loss of kidney function.


Analysis of enzyme kinetics using electrospray ionization mass spectrometry and multiple reaction monitoring: fucosyltransferase V.
An accurate, rapid, and versatile method for the analysis of enzyme kinetics using electrospray ionization mass spectrometry (ESI-MS) has been developed and demonstrated using fucosyltransferase V. Reactions performed in primary or secondary amine-containing buffers were diluted in an ESI solvent and directly analyzed without purification of the reaction products. Decreased mass resolution was used to maximize instrument sensitivity, and multiple reaction monitoring (MRM), in the tandem mass spectrometric mode, was used to enhance selectivity of detection. The approach allowed simultaneous monitoring of multiple processes, including substrate consumption, product formation, and the intensity of an internal standard. MRM gave an apparent K(m) for GDP-L-fucose (GDP-Fuc) of 50.4 +/- 5.5 microM and a k(cat) of 1.46 +/- 0.044 s(-1). Under the same conditions, the conventional radioactivity-based assay using GDP-[U-(14)C]Fuc as substrate gave virtually identical results: K(m) = 54.3 +/- 4.6 microM and k(cat) = 1.49 +/- 0.039 s(-1). The close correlation of the data showed that ESI-MS coupled to MRM is a valid approach for the analysis of enzyme kinetics. Consequently, this method represents a valuable alternative to existing analytic methods because of the option of simultaneously monitoring multiple species, the high degree of specificity, and rapid analysis times and because it does not rely on the availability of radioactive or chromogenic substrates.


Tissue-specific regulation of basic fibroblast growth factor mRNA levels by diabetes.
Because basic fibroblast growth factor (bFGF) is recognized as an angiogenic factor and diabetes is characterized by multiple vascular complications, including diabetic microangiopathy, we examined the regulation of tissue bFGF mRNA levels by diabetes. Diabetes was induced in male Sprague-Dawley rats by injection of 125 mg/kg body wt i.v. streptozocin (STZ), with intensive insulin therapy initiated in half of the diabetic rats. Rats were killed 96 h postinjection of STZ. Tissue bFGF and insulinlike growth factor I (IGF-I) mRNA levels were measured simultaneously with a solution hybridization-RNase protection assay. bFGF mRNA levels increased from 1.7- to 2.7-fold in eye, heart, lung, and brain from diabetic compared with buffer-injected control rats. In skeletal muscle, bFGF mRNA levels decreased to 23% of control levels, whereas bFGF mRNA levels were unchanged in kidneys from diabetic versus control rats. Changes in tissue bFGF mRNA levels were partially reversed by insulin treatment in all tissues. In contrast, IGF-I mRNA levels were significantly decreased from 15 to 50% of control levels in all tissues studied except those in brain, which decreased to only 85% of control levels. These data demonstrate that bFGF mRNA levels are altered by diabetes in a tissue-specific fashion and are consistent with the hypothesis that increased production of bFGF may contribute to the development of diabetic microangiopathy in some tissues.


Remodeling of ion channel expression may contribute to electrophysiological consequences caused by methamphetamine in vitro and in vivo.
Methamphetamine (MA) is a psychostimulant. MA may induce numerous cardiotoxic effects, leading to cardiac arrhythmias, heart failure, eventually leading to sudden cardiac death. The deleterious effects of methamphetamine work in tandem to disrupt the coordinated electrical activity of the heart and have been associated with life-threatening cardiac arrhythmias. Remodeling of ion channels is an important mechanism of arrhythmia. Although arrhythmogenic remodeling involves alterations in ion channel expression, it is yet unknown whether MA induced electrical remodeling by affecting gene expression, and whether the changes in protein expression are paralleled by alterations in mRNA expression. Our study focused on the expression of ion channels which were correlated to the electrical remodeling caused by MA. We used RT-PCR and western blot to assess of the transcript and translate levels of ion channel subunits, including Ito: kv1.4, kv1.7, kv3.4, kv4.2; IK1: kir2.1, kir2.2, kir2.3, kir2.4; and ICa-l: Ca(2+)α1, Ca(2+)β, respectively. The reversible effect of these changes after MA withdrawal was also evaluated. MA caused decrease in mRNA and protein levels in all ion channel subunits in vitro and also in vivo, is at this work. The kv3.4 and all 4 subunits of Kir2.0 family showed significant decrease than the other genes. Most of the channel subunit expression started to reverse after MA withdrawal for 4 weeks and significantly reverse in all of the channel subunits after MA withdrawal for 8 weeks. We found that CACNA1C and Kir2.0 family showed lower recoverability than the others after MA withdrawal for 8 weeks. The reduction of the ion channel expression levels may be the molecular mechanism that mediates the electrical remodeling caused by methamphetamine.


Direct analysis real-time-high-resolution mass spectrometry for Triticum species authentication.
Due to favourable climate condition, Italy is a prominent producer of different wheat varieties. Several wheat baked goods are produced, but the most typical Italian foods, like pasta, pizza and bread, are made of durum and common wheat flour. Because of the great importance of wheat in the Italian food market, authenticity represents an essential quality parameter not only for the producers and regulatory bodies but also for consumers. The aim of our study was to test the effectiveness of an unconventional non-targeted method for the discrimination of Triticum species using direct analysis real time-high-resolution mass spectrometry (DART-HRMS). For this purpose, 60 wheat samples including durum, common and hulled wheat varieties were collected over two consecutive harvest years. Chemometric evaluation revealed an optimal sample clustering according to the wheat species and the presence of 18 significant markers able to discriminate the groups. The discrimination power obtained is promising since the use of DART-HRMS can significantly reduce the analysis time compared to chromatographic techniques. A plausible future commercial and industrial scenario could see the application of this analytical approach especially to evaluate the risk of substitution of higher value wheat species with lower value flours.


Timing of bioprosthetic valve fracture in transcatheter valve-in-valve intervention: impact on valve durability and leaflet integrity.
Bioprosthetic valve fracture (BVF) can be used to improve transcatheter heart valve (THV) haemodynamics following a valve-in-valve (ViV) intervention. However, whether BVF should be performed before or after THV deployment and the implications on durability are unknown. Aims: We sought to assess the impact of BVF timing on long-term THV durability. The impact of BVF timing was assessed using small ACURATE neo (ACn) or 23 mm SAPIEN 3 (S3) THV deployed in 21 mm Mitroflow valves compared to no-BVF controls. Valves underwent accelerated wear testing up to 200 million (M) cycles (equivalent to 5 years). At 200M cycles, THV were evaluated by hydrodynamic testing, second-harmonic generation (SHG) microscopy, scanning electron microscopy (SEM) and histology. At 200M cycles, the regurgitant fraction (RF) and effective orifice area (EOA) for the ACn were 8.03±0.30%/1.74±0.01 cm2 (no BVF), 12.48±0.70%/1.97±0.02 cm2 (BVF before ViV) and 9.29±0.38%/2.21±0.0 cm2 (BVF after ViV), respectively. For the S3 these values were 2.63±0.51%/1.26±0.01 cm2, 2.03±0.42%/1.65±0.01 cm2, and 1.62±0.38%/2.22±0.01 cm2, respectively. Further, SHG and SEM revealed a higher degree of superficial leaflet damage when BVF was performed after ViV for the ACn and S3. However, the histological analysis revealed significantly less damage, as determined by matrix density analysis, through the entire leaflet thickness when BVF was performed after ViV with the S3 and a similar but non-significant trend with the ACn. Conclusions: BVF performed after ViV appears to offer superior long-term EOA without increased RF. Ultrastructure leaflet analysis reveals that the timing of BVF can differentially impact leaflets, with more superficial damage but greater preservation of overall leaflet structure when BVF is performed after ViV.


Primary lumbosacral Wilms tumour associated with occult spinal dysraphism.
A 4-year-old child presenting with sudden-onset paraplegia and a sacral tumour in association with spina bifida occulta is reported. There were no stigmata of spinal dysraphism at birth. Imaging studies confirmed a sacral tumour with extradural extension up to T10 and spinal dysraphism. The histological features of the extradural and sacral components of the tumour were consistent with a Wilms tumour. The differential diagnosis included a primary sacral teratoma containing Wilms tumour elements or a primary extrarenal Wilms tumour arising in association with a spinal dysraphism. There was no clinical response to chemotherapy or radiotherapy.


Association of Positive Airway Pressure Use With Acute Care Utilization and Costs.
The current value-based medical climate has led to scrutiny of all medical costs. Given the relationship between obstructive sleep apnea (OSA) and many significant comorbid medical conditions, treating patients with OSA via positive airway pressure (PAP) therapy might reduce health care expenditures. Our goal was to determine the relationship between PAP use and acute care utilization and costs. This was a retrospective cohort study of adult patients who initiated PAP therapy after a diagnosis of moderate-severe OSA at a large integrated health system during 2014-2016. The study consisted of 1,098 patients, of which 60% were on PAP > 4 h/night for ≥ 70% of the nights. The average h/night were 5.3 (standard deviation 2.4). Increasing PAP usage was associated with reduced inpatient (rate ratio [RR] 0.92, 95% confidence interval [CI] 0.86-0.98) and overall acute care visits (RR 0.96, 95% CI 0.92-0.99). The linear relationships were supported by the threshold effects identified in the categorical adherence measures. No linear association was identified with emergency department visits and inpatient stays. However, lower number of emergency department visits (RR 0.78, 95% CI 0.62-0.98) and inpatient stays (RR 0.56, 95% CI 0.35-0.91) were observed among adherent (> 4 h/night for ≥ 70% of the nights) patients. PAP usage was linearly associated with reduced number of inpatient and overall acute care visits, and lower likelihood of having positive costs from these visits. PAP usage was also associated with less emergency department visits and inpatient stays when comparing adherent patients to less adherent patients. Kirsch DB, Yang H, Maslow AL, Stolzenbach M, McCall A. Association of positive airway pressure use with acute care utilization and costs. J Clin Sleep Med. 2019;15(9):1243-1250.


T helper 1 cells and interferon gamma regulate allergic airway inflammation and mucus production.
CD4 T helper (Th) type 1 and Th2 cells have been identified in the airways of asthmatic patients. Th2 cells are believed to contribute to pathogenesis of the disease, but the role of Th1 cells is not well defined. In a mouse model, we previously reported that transferred T cell receptor-transgenic Th2 cells activated in the respiratory tract led to airway inflammation with many of the pathologic features of asthma, including airway eosinophilia and mucus production. Th1 cells caused inflammation with none of the pathology associated with asthma. In this report, we investigate the role of Th1 cells in regulating airway inflammation. When Th1 and Th2 cells are transferred together into recipient mice, there is a marked reduction in airway eosinophilia and mucus staining. To address the precise role of Th1 cells, we asked (i), Are Th2-induced responses inhibited by interferon (IFN)-gamma? and (ii) Can Th1 cells induce eosinophilia and mucus in the absence of IFN-gamma? In IFN-gamma receptor(-/-) recipient mice exposed to inhaled antigen, the inhibitory effects of Th1 cells on both airway eosinophilia and mucus production were abolished. In the absence of IFN-gamma receptor signaling, Th1 cells induced mucus but not eosinophilia. Thus, we have identified new regulatory pathways for mucus production; mucus can be induced by Th2 and non-Th2 inflammatory responses in the lung, both of which are inhibited by IFN-gamma. The blockade of eosinophilia and mucus production by IFN-gamma likely occurs through different inhibitory pathways that are activated downstream of Th2 cytokine secretion and require IFN-gamma signaling in tissue of recipient mice.


Use of trypan blue for identifying early proximal convoluted tubules.
Proximal convoluted renal tubules (PCT) are difficult to dissect for in vitro study, particularly in newborn animals, and are identified with certainty only when obtained in continuity with their parent glomeruli. We show here that the supravital colloidal dye trypan blue preferentially stains early PCT segments of rabbit in vivo and greatly facilitates their isolation. We also show that trypan blue administration does not adversely affect animal growth rate, kidney weight, or several general measures of kidney function. Using isolated PCT from newborn to adult rabbits, control (unstained) and trypan blue-stained tubules also are found not to differ in lateral membrane surface density, in their ability to maintain normal cell volume or to respond to hypotonic osmotic challenge, and in their swelling rate in isotonic medium after ouabain treatment. We conclude that trypan blue has no adverse effects on PCT cell function while greatly facilitating their isolation for physiological studies.


The influence of oxygen depletion and photosensitizer triplet-state dynamics during photodynamic therapy on accurate singlet oxygen luminescence monitoring and analysis of treatment dose response.
To date, singlet oxygen ((1)O(2)) luminescence (SOL) detection was predictive of photodynamic therapy (PDT) treatment responses both in vitro and in vivo, but accurate quantification is challenging. In particular, the early and strongest part of the time-resolved signal (500-2000ns) is difficult to separate from confounding sources of luminescence and system noise, and so is normally gated out. However, the signal dynamics change with oxygen depletion during PDT, so that this time gating biases the (1)O(2) measurements. Here, the impact of gating was investigated in detail, determining the rate constants from SOL and direct pO(2) measurements during meso-tetra(hydroxyphenyl)chlorin (mTHPC)-mediated PDT of cells in vitro under well-controlled conditions. With these data as input, numerical simulations were used to examine PDT and SOL dynamics, and the influence of various time gates on cumulative SOL signals. It is shown that gating can underestimate the SOL at early treatment time points by ∼40% and underestimate the cumulative SOL signal by 20-25%, representing significant errors. In vitro studies with both mTHPC and aminolevulinic acid-photosensitizer protoporphyrin IX demonstrate that rigorous analysis of SOL signal kinetics is then crucial in order to use SOL as an accurate and quantitative PDT dose metric.


Penetration of Nanobody-Dextran Polymer Conjugates through Tumor Spheroids.
Here we report the generation of nanobody dextran polymer conjugates (dextraknobs) that are loaded with small molecules, i.e., fluorophores or photosensitizers, for potential applications in cancer diagnostics and therapy. To this end, the molecules are conjugated to the dextran polymer which is coupled to the C-terminus of an EGFR-specific nanobody using chemoenzymatic approaches. A monovalent EGFR-targeted nanobody and biparatopic version modified with different dextran average molecular weights (1000, 5000, and 10,000) were probed for their ability to penetrate tumor spheroids. For monovalent Cy5-labeled dextraknobs, the utilization of smaller sized dextran (MW 5000 vs. 10,000) was found to be beneficial for more homogeneous penetration into A431 tumor spheroids over time. For the biparatopic dual nanobody comprising MW 1000, 5000, and 10,000 dextran labeled with photosensitizer IRDye700DX, penetration behavior was comparable to that of a direct nanobody-photosensitizer conjugate lacking a dextran scaffold. Additionally, dextraknobs labeled with IRDye700DX incubated with cells in 2D and 3D showed potent cell killing upon illumination, thus inducing photodynamic therapy (PDT). In line with previous results, monovalent nanobody conjugates displayed deeper and more homogenous penetration through spheroids than the bivalent conjugates. Importantly, the smaller size dextrans did not affect the distribution of the conjugates, thus encouraging further development of dextraknobs.


Occupancy of nonannular lipid binding sites on KcsA greatly increases the stability of the tetrameric protein.
KcsA, a homotetrameric potassium channel from prokaryotes, contains noncovalently bound lipids appearing in the X-ray crystallographic structure of the protein. The binding sites for such high-affinity lipids are referred to as "nonannular" sites, correspond to intersubunit protein domains, and bind preferentially anionic phospholipids. Here we used a thermal denaturation assay and detergent-phospholipid mixed micelles containing KcsA to study the effects of different phospholipids on protein stability. We found that anionic phospholipids stabilize greatly the tetrameric protein against irreversible, heat-induced unfolding and dissociation into subunits. This occurs in a phospholipid concentration-dependent manner, and phosphatidic acid species with acyl chain lengths ranging 14 to 18 carbon atoms are more efficient than similar phosphatidylglycerols in protecting the protein. A docking model of the KcsA-phospholipid complex suggests that the increased protein stability originates from the intersubunit nature of the binding sites and, thus, interaction of the phospholipid with such sites holds together adjacent subunits within the tetrameric protein. We also found that simpler amphiphiles, such as alkyl sulfates longer than 10 carbon atoms, also increase the protein stability to the same extent as anionic phospholipids, although at higher concentrations than the latter. Modeling the interaction of these simpler amphiphiles with KcsA and comparing it with that of anionic phospholipids serve to delineate the features of a hydrophobic pocket in the nonannular sites. Such pocket is predicted to comprise residues from the M2 transmembrane segment of a subunit and from the pore helix of the adjacent subunit and seems most relevant to protein stabilization.


Alpine Skiing With total knee ArthroPlasty (ASWAP): impact on molecular and architectural features of musculo-skeletal ageing.
This study investigated features of skeletal muscle ageing in elderly individuals having previously undergone unilateral total knee arthroplasty (TKA) and whether markers of sarcopenia could be mitigated by a 12-week alpine skiing intervention. Novel biomarkers agrin, indicative of neuromuscular junction (NMJ) degeneration, tumor suppressor protein p53, associated with muscle atrophy, and a new ultrasound-based muscle architecture biomarker were used to characterize sarcopenia. Participant details and study design are presented by Kösters et al. (2015). The results of this study show that NMJ degeneration is widespread among active septuagenarians previously subjected to TKA: all participants showed elevated agrin levels upon recruitment. At least 50% of individuals were identified as sarcopenic based on their muscle architecture, supporting the hypothesis that NMJ alterations precede sarcopenia. Notably, sarcopenia was strongly associated with the expression of p53, which seems to confirm its validity as a biomarker of muscle atrophy. Training did not significantly modify any of these biomarkers. In view of the lack of accretion of muscle mass in response to the alpine skiing intervention, we hypothesize that local muscle inflammation and oxidative stress may have blunted the anabolic response to training and promoted muscle breakdown in this elderly post-TKA population.


Lenalidomide: a brief review of its therapeutic potential in myelodysplastic syndromes.
Lenalidomide is a novel thalidomide analogue with enhanced immunomodulatory and antiangiogenic action lacking most of the typical thalidomide-associated adverse events. In myelodysplastic syndromes (MDS), it has been used primarily in the IPSS low- and intermediate-1 risk setting. Several trials have demonstrated its potential to lead to both erythroid and cytogenetic responses in these disease groups. In a clinical trial of patients with a del(5q) chromosomal abnormality, lenalidomide treatment resulted in red blood cell (RBC) transfusion independence in 67% of patients. Moreover, 45% of patients achieved a complete cytogenetic remission, and 28% achieved a minor cytogenetic remission. This result was independent of karyotype complexity. Lenalidomide might also induce long-term remissions in del(5q) patients with an elevated medullary blast count. In non-del(5q) patients, 43% of patients with confirmed low- and intermediate-1 risk achieved transfusion independence or a reduction of at least 50% of pre-treatment RBC transfusion levels. Adverse events are common but manageable and include neutropenia and thrombocytopenia, pruritus, rash, diarrhea, and others. Lenalidomide will prove an essential part in the armamentarium of MDS therapeutics. Combination therapies with cytokines, demethylating agents, tyrosine kinase inhibitors, or chemotherapy are being investigated and may show additional benefit in both low- and high risk MDS.


Fine mapping and characterization of a novel dwarf and narrow-leaf mutant dnl1 in rice.
Plant height is one of the most important agronomic traits of rice (Oryza sativa). Dwarf mutants are ideal materials for research on the mechanisms of regulation of rice plant height. We examined a new dwarf and narrow-leaf mutant dnl1. Phenotypic analysis showed that the dnl1 mutant has a thinner culm and more tillers, but the number of grains per panicle, the seed setting rate and the grain weight of dnl1 mutant were found to be significantly lower than in the wild-type. Based on scanning electron microscopic observations, the number of cells in the y-axis in internodes was significantly lower than in the wild-type. In phytohormone induction experiments, dnl1 was gibberellic acid-insensitive. The expression of some genes involved in the gibberellins metabolic pathways was affected in the dnl1 mutant, based on the real-time PCR analysis, suggesting that the dnl1 gene likely plays a role in gibberellin metabolic pathways. Genetic analysis showed that the dwarf and narrow leaf phenotype is controlled by a novel single recessive gene, here referred to as the dwarf and narrow leaf 1 (dnl1), which is located within the region between markers Ind12-11 and RM8214 on the short arm of chromosome 12. By means of fine-mapping strategy, the dnl1 gene was localized within an interval of 285.75 kb physical distance. These results will be useful for dnl1 gene cloning and to improve our understanding of the molecular mechanisms involved in the regulation of growth and development of rice.


High-affinity binding of very-long-chain fatty acyl-CoA esters to the peroxisomal non-specific lipid-transfer protein (sterol carrier protein-2).
Binding of fluorescent fatty acids to bovine liver non-specific lipid-transfer protein (nsL-TP) was assessed by measuring fluorescence resonance energy transfer (FRET) between the single tryptophan residue of nsL-TP and the fluorophore. Upon addition of pyrene dodecanoic acid (Pyr-C12) and cis-parinaric acid to nsL-TP, FRET was observed indicating that these fatty acids were accommodated in the lipid binding site closely positioned to the tryptophan residue. Substantial binding was observed only when these fatty acids were presented in the monomeric form complexed to beta-cyclodextrin. As shown by time-resolved fluorescence measurements, translocation of Pyr-C12 from the Pyr-C12-beta-cyclodextrin complex to nsL-TP changed dramatically the direct molecular environment of the pyrene moiety: i.e. the fluorescence lifetime of the directly excited pyrene increased at least by 25% and a distinct rotational correlation time of 7 ns was observed. In order to evaluate the affinity of nsL-TP for intermediates of the beta-oxidation pathway, a binding assay was developed based on the ability of fatty acyl derivatives to displace Pyr-C12 from the lipid binding site as reflected by the reduction of FRET. Hexadecanoyl-CoA and 2-hexadecenoyl-CoA were found to bind readily to nsL-TP, whereas 3-hydroxyhexadecanoyl-CoA and 3-ketohexadecanoyl-CoA bound poorly. The highest affinities were observed for the very-long-chain fatty acyl-CoA esters (24:0-CoA, 26:0-CoA) and their enoyl derivatives (24:1-CoA, 26:1-CoA). Binding of non-esterified hexadecanoic acid and tetracosanoic acid (24:0) was negligible.


Patients' experiences of the choice of GP practice pilot, 2012/2013: a mixed methods evaluation.
To investigate patients' experiences of the choice of general practitioner (GP) practice pilot. Mixed-method, cross-sectional study. Patients in the UK National Health Service (NHS) register with a general practice responsible for their primary medical care and practices set geographic boundaries. In 2012/2013, 43 volunteer general practices in four English NHS primary care trusts (PCTs) piloted a scheme allowing patients living outside practice boundaries to register as an out of area patient or be seen as a day patient. Analysis of routine data for 1108 out of area registered patients and 250 day patients; postal survey of out of area registered (315/886, 36%) and day (64/188, 34%) patients over 18 years of age, with a UK mailing address; comparison with General Practice Patient Survey (GPPS); semistructured interviews with 24 pilot patients. Pilot patients were younger and more likely to be working than non-pilot patients at the same practices and reported generally more or at least as positive experiences than patients registered at the same practices, practices in the same PCT and nationally, despite belonging to subgroups of the population who typically report poorer than average experiences. Out of area patients who joined a pilot practice did so: after moving house and not wanting to change practice (26.2%); for convenience (32.6%); as newcomers to an area who selected a practice although they lived outside its boundary (23.6%); because of dissatisfaction with their previous practice (13.9%). Day patients attended primarily on grounds of convenience (68.8%); 51.6% of the day patient visits were for acute infections, most commonly upper respiratory infections (20.4%). Sixty-six per cent of day patients received a prescription during their visit. Though the 12-month pilot was too brief to identify all costs and benefits, the scheme provided a positive experience for participating patients and practices.


[Diseases of the gastrointestinal tract and the development of stomach cancer].
An analysis of 1,108 histories of outpatients with a follow-up of 10 years and longer (up to 40 years) was carried out in Orekhovo-Zuyevo (population--132,301). 554 patients had stomach cancer diagnosed in 1973-1978 while the other 554 selected at random were in control. The results are discussed in detail.


Neonatal screening for congenital hypothyroidism by measurement of plasma thyroxine and thyroid stimulating hormone concentrations.
Neonatal screening for congenital hypothyroidism was introduced in the City of Birmingham in 1980 by measuring concentrations of both thyroid stimulating hormone and thyroxine in plasma. Over two years 30 108 babies were tested. Thirty one babies were recalled because of thyroid stimulating hormone concentrations greater than 40 mU/l, of whom 12 were treated with replacement thyroxine. Six babies were found to have low thyroxine concentrations because of reduced thyroxine binding globulin and five raised thyroxine values because of increased thyroxine binding globulin. As a result of this study screening was continued with measurement of thyroid stimulating hormone only as the primary test for congenital hypothyroidism, the thyroxine value being measured only when the concentration of thyroid stimulating hormone exceeded 20 mU/l.


Drug Transport by the Blood-Aqueous Humor Barrier of the Eye.
The ocular barriers (cornea, blood-retinal barrier, and blood-aqueous humor barrier) make treating eye diseases with therapeutic drugs challenging. The tight capillary endothelium of the iris and the ciliary body epithelium form the blood-aqueous humor barrier. The iris and ciliary body (iris-ciliary body) express a variety of drug transporters in the ATP-binding cassette and solute carrier (SLC) families. ATP-binding cassette family drug transporters that are present in the iris-ciliary body include P-glycoprotein, breast cancer resistance protein, and several multidrug resistance-associated proteins. SLC family drug transporters that are present in the iris-ciliary body include organic anion transporters, organic anion transporting polypeptides, bile acid transporters (apical sodium-dependent bile salt transporter and sodium taurocholate cotransporter), organic cation transporters (novel organic cation transporter and multidrug and toxin extrusion transporter) and peptide transporters. Freshly dissected iris-ciliary body preparations actively accumulate a variety of substrates of SLC drug transporters that are expressed in the tissue. The ciliary body in vitro supports active transport in the aqueous humor-to-blood direction of several substrates of organic anion transporters and multidrug resistance-associated proteins, consistent with the subcellular localization of these transporters in the ciliary body epithelium. In vivo data suggest that drug transporters in the iris-ciliary body reduce the permeation of drugs in the direction of blood-to-aqueous humor, thereby reducing ocular drug bioavailability, and are also involved in active drug elimination from the aqueous humor. An understanding of the influence on pharmacokinetics of drug transporters in the blood-aqueous humor barrier should help improve drug delivery and efficacy in the eye.


Inhibition of TLR9-NF-κB-mediated sterile inflammation improves pressure overload-induced right ventricular dysfunction in rats.
Recent accumulating evidence suggests that sterile inflammation plays a crucial role in the progression of various cardiovascular diseases. However, its contribution to right ventricular (RV) dysfunction remains unknown. The aim of this study was to elucidate whether toll-like receptor 9 (TLR9)-NF-κB-mediated sterile inflammation plays a critical role in the pathogenesis of RV dysfunction. We performed main pulmonary artery banding (PAB) in rats to induce RV pressure overload and dysfunction. On Day 14 after PAB, the pressure overload impaired RV function as indicated by increased RV end-diastolic pressure concomitant with macrophage infiltration and fibrosis, as well as maximal activation of NF-κB and TLR9. Short-term administration (days 14-16 after PAB) of a specific TLR9 inhibitor, E6446, or an NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC) significantly attenuated NF-κB activation. Furthermore, long-term administration of E6446 (treatment: days 14-28) or PDTC (prevention: days -1 to 28; treatment: days 14 to 28) improved RV dysfunction associated with mitigated macrophage infiltration and fibrosis in right ventricle and decreased serum brain natriuretic peptide levels. Inhibition of TLR9-NF-κB pathway-mediated sterile inflammation improved PAB-induced RV dysfunction in rats. This pathway plays a major role in the progression of pressure overload-induced RV dysfunction and is potentially a novel therapeutic target for the disorder.


Changes in subchondral bone in cartilage resurfacing--an experimental study in sheep using different types of osteochondral grafts.
This article addresses the subchondral bone integrity in cartilage resurfacing by comparing fresh, untreated auto-, xeno-, and photooxidized osteochondral allo- and xenografts. Photooxidation was expected to improve mechanical stability of the osteochondral grafts through an improved linkage of the collagen fibers within the bone matrix. Untreated auto- and xenografts and with photooxidation pretreated allo- and xenografts were surgically implanted in femoral condyles of sheep (n=40). After 2, 6, 12 and 18 months results were evaluated histologically using non-decalcified bone embedded in acrylic resin. Qualitative evaluation was performed with emphasis on bone matrix, biomechanical stability of graft anchorage, formation of cystic lesions, and bone resorption and formation. Quantitative evaluation of the total subchondral bone area was conducted histomorphometrically. Statistical analysis (factorial ANOVA test) was used to compare differences between groups with respect to the percentage of bone matrix and fibrous tissue per section. Subchondral bone resorption was fastest in untreated, fresh autografts, followed by photooxidized allografts, untreated, fresh xenografts and last pretreated photooxidized xenografts. Cystic lesions were seen in all types of grafts, but were most pronounced at 6 months in autografts and least in photooxidized grafts. Cyst-like lesions had subsided substantially in the untreated auto- and photooxidized xenografts, if no graft dislocation occurred during the healing period. Mononuclear cell infiltration and an increase in the presence of multinuclear cells were observed at 2 months, mostly in untreated autografts, followed by photooxidized allo- and untreated xenografts. They were much higher in numbers compared to photooxidized grafts, at least in the early specimens at 2 months. Graft stability was linked to the rate of bone resorption. Substantial resorption of the subchondral bone, involving the development of cyst-like lesions, lead to dislocation and finally to cartilage matrix degradation of the grafts. The process of photooxidation decreased the speed of bone resorption in osteochondral grafts and, thus, improved graft stability and cartilage survival. These results suggest that the remodeling of the subchondral bone of the host and the graft within the first 6 months is an important factor in graft stability and overall results of cartilage resurfacing.