S4 Segment Research Articles

Immunohistochemical Localization of Alogliptin, a DPP-4 Inhibitor, in Tissues of Normal and Type 2 Diabetes Model Rat.

We investigated the pharmacokinetics of alogliptin (AG) at the cell and tissue level in healthy Wistar rats and a type 2 diabetic Goto-Kakizaki (GK) rat model. Immunohistochemistry of the renal tissue in these rats, post 1 hr of AG administration, showed that the signal was observed in the glomeruli, proximal tubule S3 segments, distal tubules, collecting ducts, and only in the brush border of the epithelial cells of the proximal tubule S1, S2 segments. After 6 hr of AG administration, the staining intensity of the regions other than the S3 segments was considerably reduced in Wistar rats, with no change observed in GK rats. At 24 hr, the staining intensity was considerably reduced, even in GK rats; however, the staining of the S3 segment remained unaltered in both. Hepatocytes in zone III of the hepatic lobule were more intensely stained than those in zone I in Wistar rats at 1 hr. However, almost no staining was observed in the hepatocytes of GK rats at 1 hr. Complete loss of signal was observed in the hepatocytes of the Wistar rats after 6 hr. This study revealed that the pharmacokinetics of AG in GK rats are different from those in Wistar rats.

Detection and phylogenetic assessment of PRV-1 via sampling of biological materials released from salmon farms in British Columbia

The growth of the net pen salmon farming industry and the concurrent decline of many wild salmon populations have generated an ongoing call for research into the potential role of infectious agents associated with salmon farming. Here, we describe a method to screen for the presence of viral nucleic acids by sampling biological waste in the vicinity of farms. We collected biological samples adjacent to 56 marine net pens from five different companies, and two farm salmon processing plants ( n = 230), 70% of samples were positive for Piscine orthoreovirus-1 (PRV-1). Viral genome segments S1 ( n = 68) and M2 ( n = 39) were sequenced and subjected to phylogenetic analysis. Sequences were all monophyletic to the clade of PRV-1 routinely found in the region. Consistent with previous studies, samples collected near Atlantic salmon ( Salmo salar) farms were related to a lineage of PRV-1a commonly detected in both farmed and wild salmon in British Columbia, while a related lineage of PRV-1a was detected near Chinook salmon ( Oncorhynchus tshawytscha) farms. Similarity in sequence within companies suggests that Atlantic salmon freshwater hatcheries are a probable source of this virus to the marine environment, concordant with recent detections of PRV-1 within those hatcheries.

Finite Element Analysis and Transiliac-Transsacral Screw Fixation for Posterior Pelvic Ring with Sacrum Dysplasia.

Posterior pelvic ring sacroiliac screws are preferred by clinicians for their good biomechanical performance. However, there are few studies on mechanical analysis and intraoperative screw insertion of the dysplastic sacrum and sacroiliac screw. This study investigated the biomechanical performance of oblique sacroiliac screws (OSS) in S1 combined with transiliac-transsacral screws (TTSs) in S2 for pelvic fracture or sacroiliac dislocation with dysplastic sacrum and evaluated the safety of screw placement assisted by the navigation template. Six models were established, including one OSS fixation in the S2 segment, one transverse sacroiliac screw (TSS) fixation in the S2 segment, one TTS fixation in the S2 segment, one OSS fixation in the S1 and S2 segments, one OSS fixation in the S1 segment and one TSS fixation in the S2 segment, one OSS fixation in the S1 segment and one TTS fixation in the S2 segment. Then, finite element analysis (FEA) was performed. Twelve dysplastic sacrum patients with pelvis fracture or sacroiliac dislocation underwent OSS insertion in the S1 combined with TTS insertion in the S2 under the assistance of the patient-specific locked navigation template. Grading and Matta scores were evaluated after surgery. In the one-screw fixation group, the vertical displacements of the sacrum surface of S2 OSS, S2 TSS and S2 TTS were 1.23, 1.42, and 1.22 mm, respectively, and the maximum stress of screw were 139.45 MPa, 144.81 MPa, 126.14 MPa, respectively. In the two-screw fixation group, the vertical displacements of the sacrum surface of the S1 OSS + S2 OSS, S1 OSS + S2 TSS and S1 OSS + S2 TTS were 0.91, 1.06, and 0.75 mm, respectively, and the maximum stress of screw were 149.26 MPa, 167.13 Pa, 136.76 MPa, respectively. Clinically, a total of 12 TTS and OSS were inserted under the assistance of navigation templates, with a surgical time of 55 ± 7.69 min, bleeding of 57.5 ± 18.15 ml and radiation times of 14.5 ± 4.95. One of the TTS and one of the OSS were grade 1, and the other screws were grade 0. The Matta scores of nine patients were excellent, and three patents were good. OSS in the S1 combined with TTS in the S2 had the best mechanical stability in six models, and it is safe for screw insertion assisted by the patient-specific locked navigation template.

Robot-assisted versus navigation-assisted screw placement in spinal vertebrae

PurposeBoth robots and navigation are effective strategies for optimizing screw placement, as compared to freehand placement. However, few studies have compared the accuracy and efficiency of these two techniques. Thus, the purpose of this study is to compare the accuracy and efficiency of robotic and navigation-assisted screw placement in the spinal vertebrae.MethodsThe 24 spine models were divided into a robot- and navigation-assisted groups according to the left and right sides of the pedicle. The C-arm transmits image data simultaneously to the robot and navigates using only one scan. After screw placement, the accuracy of the two techniques were compared using “angular deviation” and “Gertzbein and Robbins scale” in different segments (C1–7, T1–4, T5–8, T9–12, and L1–S1). In addition, operation times were compared between robot- and navigation-assisted groups.ResultsRobots and navigation systems can simultaneously assist in screw placement. The robot-assisted group had significantly less angular deviation than the navigation-assisted group from C1 to S1 (p < 0.001). At the C1–7 and T1–4 segments, the robot-assisted group had a higher rate of acceptable screws than the robot-assisted group. However, at the T5–8, T9–12, and L1–S1 segments, no significant difference was found in the incidence of acceptable screws between the two groups. Moreover, robot-assisted screw placement required less operative time than navigation (p < 0.05).ConclusionThe robot is more accurate and efficient than navigation in aiding screw placement. In addition, robots and navigation can be combined without increasing the number of fluoroscopic views.

Genetic characterization of a novel pheasant-origin orthoreovirus using Next-Generation Sequencing.

A field isolate (Reo/SDWF /Pheasant/17608/20) of avian orthoreovirus (ARV), isolated from a flock of game-pheasants in Weifang, Shandong Province, was genetically characterized being a field variant or novel strain in our recent research studies in conducting whole genome sequencing by using Next-Generation Sequencing (NGS) technique on Illumina MiSeq platform. Among a total of 870,197 35-151-mer sequencing reads, 297,711 reads (34.21%) were identified as ARV sequences. The de novo assembly of the ARV reads resulted in generation of 10 ARV-related contigs with the average sequencing coverage from 1390× to 1977× according to 10 ARV genome segments. The complete genomes of this pheasant-origin ARV (Reo/SDWF /Pheasant/17608/20) were 23,495 bp in length and consist of 10 dsRNA segments ranged from 1192 bp (S4) to 3958 bp (L1) encoding 12 viral proteins. Sequence comparison between the SDWF17608 and classic ARV reference strains revealed that 58.1-100% nucleotide (nt) identities and 51.4-100% amino acid (aa) identities were in genome segment coding genes. The 10 RNA segments had conversed termini at 5' (5'-GCUUUU) and 3' (UCAUC-3') side, which were identical to the most published ARV strains. Phylogenetic analysis revealed that this pheasant ARV field variant was closely related with chicken ARV strains in 7 genome segment genes, but it possessed significant sequence divergence in M1, M3 and S2 segments. These findings suggested that this pheasant-origin field variant was a divergent ARV strain and was likely originated from reassortments between different chicken ARV strains.

Gating intermediates reveal inhibitory role of the voltage sensor in a cyclic nucleotide-modulated ion channel

Understanding how ion channels gate is important for elucidating their physiological roles and targeting them in pathophysiological states. Here, we used SthK, a cyclic nucleotide-modulated channel from Spirochaeta thermophila, to define a ligand-gating trajectory that includes multiple on-pathway intermediates. cAMP is a poor partial agonist for SthK and depolarization increases SthK activity. Tuning the energy landscape by gain-of-function mutations in the voltage sensor domain (VSD) allowed us to capture multiple intermediates along the ligand-activation pathway, highlighting the allosteric linkage between VSD, cyclic nucleotide-binding (CNBD) and pore domains. Small, lateral displacements of the VSD S4 segment were necessary to open the intracellular gate, pointing to an inhibitory VSD at rest. We propose that in wild-type SthK, depolarization leads to such VSD displacements resulting in release of inhibition. In summary, we report conformational transitions along the activation pathway that reveal allosteric couplings between key sites integrating to open the intracellular gate.

Optimized tight binding between the S1 segment and KCNE3 is required for the constitutively open nature of the KCNQ1-KCNE3 channel complex.

Tetrameric voltage-gated K⁺ channels have four identical voltage sensor domains, and they regulate channel gating. KCNQ1 (Kv7.1) is a voltage-gated K⁺ channel, and its auxiliary subunit KCNE proteins dramatically regulate its gating. For example, KCNE3 makes KCNQ1 a constitutively open channel at physiological voltages by affecting the voltage sensor movement. However, how KCNE proteins regulate the voltage sensor domain is largely unknown. In this study, by utilizing the KCNQ1-KCNE3-calmodulin complex structure, we thoroughly surveyed amino acid residues on KCNE3 and the S1 segment of the KCNQ1 voltage sensor facing each other. By changing the side-chain bulkiness of these interacting amino acid residues (volume scanning), we found that the distance between the S1 segment and KCNE3 is elaborately optimized to achieve the constitutive activity. In addition, we identified two pairs of KCNQ1 and KCNE3 mutants that partially restored constitutive activity by co-expression. Our work suggests that tight binding of the S1 segment and KCNE3 is crucial for controlling the voltage sensor domains.

Breakage risk of different sacroiliac screws in unilateral sacral fractures a finite element analysis

ObjectiveTo compare the breakage risk of lengthened sacroiliac screws and ordinary sacroiliac screws to treat unilateral vertical sacral fractures and provide a reference for clinical application.MethodsA finite element model of Tile C pelvic ring injury (unilateral type Denis II fracture of the sacrum) was produced. The sacral fractures were fixed with a lengthened sacroiliac screw and ordinary sacroiliac screw in 6 types of models. The maximal von Mises stresses and stress distributions of the two kinds of screws when standing on both feet were measured and compared.ResultsThe maximal von Mises stress of the lengthened screw was less than that of the ordinary screw. Compared with ordinary screw, the stress distribution in the lengthened screw was more homogeneous.ConclusionsThe breakage risk of screws fixed in double segments is lower than that of screws fixed in single segments, the breakage risk of lengthened screws is lower than that of ordinary screws, and the breakage risk of screws fixed in S2 segments is lower than that of screws fixed in S1 segments.

Chromosome-scale assemblies of the male and female Populus euphratica genomes reveal the molecular basis of sex determination and sexual dimorphism

Reference-quality genomes of both sexes are essential for studying sex determination and sex-chromosome evolution, as their gene contents and expression profiles differ. Here, we present independent chromosome-level genome assemblies for the female (XX) and male (XY) genomes of desert poplar (Populus euphratica), resolving a 22.7-Mb X and 24.8-Mb Y chromosome. We also identified a relatively complete 761-kb sex-linked region (SLR) in the peritelomeric region on chromosome 14 (Y). Within the SLR, recombination around the partial repeats for the feminizing factor ARR17 (ARABIDOPSIS RESPONSE REGULATOR 17) was potentially suppressed by flanking palindromic arms and the dense accumulation of retrotransposons. The inverted small segments S1 and S2 of ARR17 exhibited relaxed selective pressure and triggered sex determination by generating 24-nt small interfering RNAs that induce male-specific hyper-methylation at the promoter of the autosomal targeted ARR17. We also detected two male-specific fusion genes encoding proteins with NB-ARC domains at the breakpoint region of an inversion in the SLR that may be responsible for the observed sexual dimorphism in immune responses. Our results show that the SLR appears to follow proposed evolutionary dynamics for sex chromosomes and advance our understanding of sex determination and the evolution of sex chromosomes in Populus.

The 6 degrees-of-freedom range of motion of the L1-S1 vertebrae in young and middle-aged asymptomatic people.

Controlled laboratory study. To determine the 6 degrees of freedom of lumbar vertebra in vivo during different functional activities in young and middle-aged asymptomatic subjects. A total of 26 asymptomatic subjects (M/F, 15/11; age, 20-55 years) were recruited in this study. They were divided into two groups: young group (number: 14; age: 20-30 years old) and middle-aged group (number: 12; age: 45-55 years old). The lumbar segment of each subject was scanned by computed tomography for the construction of three-dimensional (3D) models of the vertebra from L1 to S1. The lumbar spine was imaged by using a dual fluoroscopic system when the subjects performed different trunk postures. The 3D models of vertebrae were matched to two fluoroscopic images simultaneously in software. The range of motion (ROM) of vertebrae in the young and middle-aged groups was compared by using multiway analysis of variance, respectively. During the supine to the upright posture, vertebral rotation of the L1-S1 occurred mainly around the mediolateral axis (mean: 3.9 ± 2.9°). Along the mediolateral axis, vertebral translation was significantly lower at L1-2 (7.7 ± 2.4 mm) and L2-3 (8.0 ± 3.5 mm) than at L3-4 (1.6 ± 1.2 mm), L4-5 (3.3 ± 2.6 mm), and L5-S1 (2.6 ± 1.9 mm). At the L4-5 level, the young group had a higher rotational ROM than the middle-aged group around all three axes during left-right bending. Along the anteroposterior axis, the young group had a lower translational ROM at L4-5 than the middle-aged group during left-right bending (4.6 ± 3.3 vs. 7.6 ± 4.8 mm; P < 0.05). At L5-S1, the young group had a lower translational ROM than the middle-aged group during flexion-extension, left-right bending, and left-right torsion. This study explored the lumbar vertebral ROM at L1-S1 during different functional postures in both young and middle-aged volunteers. There were higher coupled translations at L3-4 and L4-5 than at the upper lumbar segments during supine to upright. The vertebral rotation decreased with age. In addition, the older subjects had a higher anteroposterior translation at the L4-5 segment and higher mediolateral translation at the L5-S1 segment than the young group. These data might provide basic data to be compared with spinal pathology.

Mechanism of voltage gating in the voltage-sensing phosphatase Ci-VSP

Conformational changes in voltage-sensing domains (VSDs) are driven by the transmembrane electric field acting on the protein charges. Yet, the overall energetics and detailed mechanism of this process are not fully understood. Here, we determined free energy and displacement charge landscapes as well as the major conformations visited during a complete functional gating cycle in the isolated VSD of the phosphatase Ci-VSP (Ci-VSD) comprising four transmembrane helices (segments S1 to S4). Molecular dynamics simulations highlight the extent of S4 movements. In addition to the crystallographically determined activated "Up" and resting "Down" states, the simulations predict two Ci-VSD conformations: a deeper resting state ("down-minus") and an extended activated ("up-plus") state. These additional conformations were experimentally probed via systematic cysteine mutagenesis with metal-ion bridges and the engineering of proton conducting mutants at hyperpolarizing voltages. The present results show that these four states are visited sequentially in a stepwise manner during voltage activation, each step translocating one arginine or the equivalent of ∼1 e0 across the membrane electric field, yielding a transfer of ∼3 e0 charges in total for the complete process.

The role of Phe150 in human voltage-gated proton channel

The voltage-gated proton channel Hv1 is a member of voltage-gated ion channels containing voltage-sensing domains (VSDs). The VSDs are made of four membrane-spanning segments (S1 through S4), and their function is to detect changes in membrane potential in the cells. A highly conserved phenylalanine 150 (F150) is located in the S2 segment of human voltage-gated proton channels. We previously discovered that the F150 is a binding site for the open channel blocker 2GBI. Here, we show that the Hv1 VSD voltage-dependent activation requires a hydrophobic group at position F150. We perform double-mutant cycle analysis to probe interactions between F150 and positively charged arginines in the S4 segment of the channel. Our results indicate that F150 interacts with two arginines (R2 and R3) in the S4 segment and catalyzes the transfer of the S4 arginines in the process of voltage-dependent activation.

A novel de novo KCNB1 variant altering channel characteristics in a patient with periventricular heterotopia, abnormal corpus callosum, and mild seizure outcome.

KCNB1 encodes the α-subunit of Kv2.1, the main contributor to neuronal delayed rectifier potassium currents. The subunit consists of six transmembrane α helices (S1-S6), comprising the voltage-sensing domain (S1-S4) and the pore domain (S5-P-S6). Heterozygous KCNB1 pathogenic variants are associated with developmental and epileptic encephalopathy. Here we report an individual who shows the milder phenotype compared to the previously reported cases, including delayed language development, mild intellectual disability, attention deficit hyperactivity disorder, late-onset epilepsy responsive to an antiepileptic drug, elevation of serum creatine kinase, and peripheral axonal neuropathy. On the other hand, his brain MRI showed characteristic findings including periventricular heterotopia, polymicrogyria, and abnormal corpus callosum. Exome sequencing identified a novel de novo KCNB1 variant c.574G>A, p.(Ala192Thr) located in the S1 segment of the voltage-sensing domain. Functional analysis using the whole-cell patch-clamp technique in Neuro2a cells showed that the Ala192Thr mutant reduces both activation and inactivation of the channel at membrane voltages in the range of -50 to -30 mV. Our case could expand the phenotypic spectrum of patients with KCNB1 variants, and suggested that variants located in the S1 segment might be associated with a milder outcome of seizures.

Development of immunohistochemistry for detecting fluvoxamine in rat tissues using newly prepared monoclonal antibody: its precise localization in small intestine, kidney, and liver of rats.

A monoclonal antibody (mAb) was produced against a fluvoxamine (FLV)-bovine serum albumin conjugate that was specific to both the conjugate and free form of FLV. The mAb enabled us to develop an immunohistochemistry (IHC) method for pharmacokinetic analysis of FLV at the cell and tissue levels. We demonstrated that IHC can be used to detect the localization of FLV in the small intestine, kidney, and liver 1 h after drug administration at the cell and tissue levels. Protease digestion is an important factor for obtaining appropriate IHC staining results for localization of drugs. In this study, precise FLV localization could be determined with only 1 h of protease digestion in the kidneys, but in the small intestine and liver, the staining results with two digestive conditions had to be merged. IHC provided new findings, such as (1) nerve cells are likely to uptake more FLV than other cells and tissues; (2) the ability of reabsorption and secretion in the kidney varies depending on the site, and the amount of FLV in the primary urine is regulated downstream of the proximal tubule S3 segment; and (3) some of the FLV is excreted in the bile.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00795-022-00337-6.

Thoracoscopic S2 segmentectomy by a posterior approach for a central metastasis: a case report

: Central metastases are an ideal indication for anatomic sublobar resection. This allows adequate, safe resection, with preservation of lung function and decreased morbidity. This paper illustrates a case of S2 segmentectomy for a central metastasis and highlights key technical considerations. The author finds that a direct posterior approach to S2 facilitates the operation, minimizing dissection and allowing sparing of the horizontal fissure which may reduce the potential for prolonged air leak. Sparing tissue planes may also facilitate redo-surgery and increase therapeutic options in the event of future metastatic disease. Anatomic sublobar resection requires attention to detail. Planning is very important, and advanced imaging including 3D reconstruction and 3D printing is increasingly being considered for this purpose. Intraoperatively, correct positioning of trocars and deliberate, purposeful retraction are key. Finally, a challenge specific to the resection of central S2 segment lesions is the requirement for sufficient space at the base of the segment to allow parenchymal division with negative margins. Methodical identification, dissection, and division of the segmental venous drainage is an important step that allows the space at the base of the segment to open up. This space can then be dissected widely in order to position the stapler and complete the resection, even for lesions at the very root of the segment.

Open channel block of Kv1.5 channels by HMQ1611.

Kv1.5 channels conduct the ultra-rapid delayed rectifier potassium current (I Kur). Pharmacological blockade of human Kv1.5 (hKv1.5) has been regarded as an effective treatment of re-entrant based atrial fibrillation, because Kv1.5 is highly expressed in human cardiac atria but scarcely in ventricles. The Kv1.5 blockade is also expected to be used in cancer therapeutics since Kv1.5 is overexpressed in some types of human tumors. Here, we investigated the blockade of hKv1.5 channels by HMQ1611, a symmetrical biphenyl derivative. hKv1.5 channels were heterologously expressed in Chinese hamster ovary cells. The effects of HMQ1611 on wild-type and 13 hKv1.5 mutant channels were examined using the whole-cell patch-clamp method, and molecular docking simulation was conducted to predict the docking position of HMQ1611 within Kv1.5 channels. We showed that HMQ1611 reversibly inhibited the hKv1.5 current in a concentration-dependent manner (IC50 = 2.07 μM). HMQ1611 blockade of hKv1.5 current developed with time during depolarizing voltage-clamp steps, and this blockade was also voltage-dependent with a steep increase over the voltage range for channel openings. HMQ1611 inhibition was significantly reduced in the T479A, T480A, V505A, I508A, L510A, V512A, and V516A hKv1.5 mutant channels. Molecular docking analysis predicted that V505, V512, and T480 were involved in the blocking action of HMQ1611 on hKv1.5 channels. These results suggest that HMQ1611 inhibits hKv1.5 currents as an open channel blocker. Amino acid residues located at the base of the selectivity filter (T479 and T480) and in the S6 segment (V505, I508, L510, V512, and V516) of hKv1.5 appear to constitute potential binding sites for HMQ1611.

A TRPM7 mutation linked to familial trigeminal neuralgia: Omega current and hyperexcitability of trigeminal ganglion neurons

Trigeminal neuralgia (TN) is a unique pain disorder characterized by intense paroxysmal facial pain within areas innervated by the trigeminal nerve. Although most cases of TN are sporadic, familial clusters of TN suggest that genetic factors may contribute to this disorder. Whole-exome sequencing in patients with TN reporting positive family history demonstrated a spectrum of variants of ion channels including TRP channels. Here, we used patch-clamp analysis and Ca2+ and Na+ imaging to assess a rare variant in the TRPM7 channel, p.Ala931Thr, within transmembrane domain 3, identified in a man suffering from unilateral TN. We showed that A931T produced an abnormal inward current carried by Na+ and insensitive to the pore blocker Gd3+. Hypothesizing that replacement of the hydrophobic alanine at position 931 with the more polar threonine destabilizes a hydrophobic ring, near the voltage sensor domain, we performed alanine substitutions of F971 and W972 and obtained results suggesting a role of A931-W972 hydrophobic interaction in S3-S4 hydrophobic cleft stability. Finally, we transfected trigeminal ganglion neurons with A931T channels and observed that expression of this TRPM7 variant lowers current threshold and resting membrane potential, and increases evoked firing activity in TG neurons. Our results support the notion that the TRPM7-A931T mutation located in the S3 segment at the interface with the transmembrane region S4, generates an omega current that carries Na+ influx in physiological conditions. A931T produces hyperexcitability and a sustained Na+ influx in trigeminal ganglion neurons that may underlie pain in this kindred with trigeminal neuralgia.

An Adaptable Physiological Model of Endocytic Megalin Trafficking in Opossum Kidney Cells and Mouse Kidney Proximal Tubule.

The cells that comprise the proximal tubule (PT) are specialized for high-capacity apical endocytosis necessary to maintain a protein-free urine. Filtered proteins are reclaimed via receptor-mediated endocytosis facilitated by the multiligand receptors megalin and cubilin. Despite the importance of this pathway, we lack a detailed understanding of megalin trafficking kinetics and how they are regulated. Here, we utilized biochemical and quantitative imaging methods in a highly differentiated model of opossum kidney (OK) cells and in mouse kidney in vivo to develop mathematical models of megalin traffic. A preliminary model based on biochemically quantified kinetic parameters was refined by colocalization of megalin with individual apical endocytic compartment markers. Our model predicts that megalin is rapidly internalized, resulting in primarily intracellular distribution of the receptor at steady state. Moreover, our data show that early endosomes mature rapidly in PT cells and suggest that Rab11 is the primary mediator of apical recycling of megalin from maturing endocytic compartments. Apical recycling represents the rate-limiting component of endocytic traffic, suggesting that this step has the largest impact in determining the endocytic capacity of PT cells. Adaptation of our model to the S1 segment of mouse PT using colocalization data obtained in kidney sections confirms basic aspects of our model and suggests that our OK cell model largely recapitulates in vivo membrane trafficking kinetics. We provide a downloadable application that can be used to adapt our working parameters to further study how endocytic capacity of PT cells may be altered under normal and disease conditions.

Cholesterol inhibits human voltage-gated proton channel hHv1

Although human sperm is morphologically mature in the epididymis, it cannot fertilize eggs before capacitation. Cholesterol efflux from the sperm plasma membrane is a key molecular event essential for cytoplasmic alkalinization and hyperactivation, but the underlying mechanism remains unclear. The human voltage-gated proton (hHv1) channel functions as an acid extruder to regulate intracellular pHs of many cell types, including sperm. Aside from voltage and pH, Hv channels are also regulated by distinct ligands, such as Zn2+ and albumin. In the present work, we identified cholesterol as an inhibitory ligand of the hHv1 channel and further investigated the underlying mechanism using the single-molecule fluorescence resonance energy transfer (smFRET) approach. Our results indicated that cholesterol inhibits the hHv1 channel by stabilizing the voltage-sensing S4 segment at resting conformations, a similar mechanism also utilized by Zn2+. Our results suggested that the S4 segment is the central gating machinery in the hHv1 channel, on which voltage and distinct ligands are converged to regulate channel function. Identification of membrane cholesterol as an inhibitory ligand provides a mechanism by which the hHv1 channel regulates fertilization by linking the cholesterol efflux with cytoplasmic alkalinization, a change that triggers calcium influx through the CatSper channel. These events finally lead to hyperactivation, a remarkable change in the mobility pattern indicating fertilization competence of human sperm.

Living-donor segmental lung transplantation for pediatric patients

ObjectiveThe preset study evaluated the outcome of living-donor segmental lung transplantation for pediatric patients. MethodsBetween August 2009 and May 2021, we performed living-donor segmental lung transplantation in 6 critically ill pediatric patients, including 1 patient on a ventilator alone and another patient on a ventilator and extracorporeal membrane oxygenation (ECMO). There were 4 male and 2 female patients, with a median age of 7 years (range, 4-15 years) and a median height of 112.7 cm (range, 95-125.2 cm). The diagnoses included complications of allogeneic hematopoietic stem cell transplantation (n = 4) and pulmonary fibrosis (n = 2). All patients received bilateral lung transplantation under cardiopulmonary bypass. A basal segment and a lower lobe were implanted in 3 patients, and a basal segment and an S6 segment were implanted in the other 3 patients. In 2 patients, the right S6 segmental graft was horizontally rotated 180° and implanted as the left lung. ResultsAmong the 9 segmental grafts implanted, 7 functioned well after reperfusion. Two rotated S6 segmental grafts became congestive, with 1 requiring graft extraction and the other venous repair, which was successful. There was 1 hospital death (14 days) due to sepsis and 1 late death (9 years) due to leukoencephalopathy. The remaining 4 patients are currently alive at 9 months, 10 months, 1.3 years, and 1.9 years. ConclusionsLiving-donor segmental lung transplantation was a technically difficult but feasible procedure with acceptable outcomes for small pediatric patients with chest cavities that were too small for adult lower lobe implantation.