CGRP Released by Corneal Sensory Nerve Maintains Tear Secretion of the Lacrimal Gland.

Purpose: This study aims to elucidate the calcitonin gene-related peptide (CGRP) mediation and primary mechanism of corneal sensory nerves on tear production of the lacrimal gland. Methods: Mouse corneal denervation models were constructed through surgical axotomy, pharmacologic treatment with capsaicin or resiniferatoxin, and Trpv1-Cre/DTR mice with diphtheria toxin injection. The capsaicin-treated mice received subconjunctival injection of CGRP or substance P, while the normal C57BL/6J mice were administered with CGRP receptor antagonist BIBN-4096. Furthermore, double immunostaining of c-FOS+ and choline acetyltransferase was used to evaluate the activation of the superior salivatory nucleus (SSN). Mouse lacrimal glands were collected for transcriptomic sequencing and subsequent RNA and protein expression analysis. Results: The corneal denervated mice exhibited a significant reduction in corneal sensitivity and tear secretion. In capsaicin-treated mice, tear secretion decreased to 2.5 ± 0.5 mm compared to 6.3 ± 0.9 mm in control mice (P < 0.0001). However, exogenous administration of CGRP in capsaicin-treated mice increased tear secretion from 2.6 ± 0.5 mm to 4.5 ± 0.5 mm (P = 0.0009), while BIBN-4096 treatment reduced tear secretion to 3.4 ± 0.5 mm when compared to 7.3 ± 0.7 mm in control mice (P = 0.0022). Furthermore, c-FOS+ cell number in the SSN increased by twofold (P = 0.0168) after CGRP administration compared with capsaicin-treated mice. In addition, the expressions of CCNA2, Ki67, PCNA, and CDK1 in acinar cells of the lacrimal gland were impaired by corneal denervation and alleviated by CGRP administration. Conclusions: CGRP released by corneal sensory nerves mediates tear secretion of the lacrimal gland, providing a new strategy for improving tear secretion in patients with neurotrophic keratitis.

Final phase I substudy results of ivosidenib in patients with mutant IDH1 relapsed/refractory myelodysplastic syndrome.

Ivosidenib is a first-in-class mutant isocitrate dehydrogenase 1 (mIDH1) inhibitor and has shown efficacy and tolerability in patients with advanced mIDH1 hematologic malignancies, leading to approval in front-line and relapsed/refractory (R/R) mIDH1 AML populations. We report final data from a phase I single-arm substudy (NCT02074839) of patients with R/R mIDH1 MDS following failure of standard-of-care therapies. Oral ivosidenib was taken once daily on days 1-28 in 28-day cycles. Primary objectives were to determine safety, tolerability, and clinical activity. The primary efficacy endpoint was the complete remission + partial remission (CR+PR) rate. Nineteen patients were enrolled; 18 were included in the efficacy analysis. Treatment-related adverse events occurred in eight (42.1%) patients, including a grade 1 QT interval prolongation in one (5.3%) patient and grade 2 differentiation syndrome in two (10.5%) patients. Rates of CR+PR and objective response (CR +PR+marrow CR) were 38.9% (95% confidence interval [CI]: 17.3, 64.3) and 83.3% (95% CI: 58.6, 96.4), respectively. Kaplan-Meier estimates showed a 68.6% probability of patients in CR achieving a remission duration of >=5 years, and a median OS of 35.7 months. Of note, 71.4% and 75.0% baseline red blood cell (RBC) and platelet transfusion-dependent patients, respectively, became transfusion independent (TI; no transfusion >=56 days); 81.8% and 100% of baseline RBC and platelet TI patients, respectively, remained TI. One (5.3%) patient proceeded to a hematopoietic stem cell transplant by data cut-off. In conclusion, ivosidenib is clinically active, with durable remissions and a manageable safety profile observed in patients with mIDH1 R/R MDS.

cGAS/STING signaling aggravated corneal allograft rejection through neutrophil extracellular traps (NETs).

The activation of innate immunity following transplantation has been identified as a crucial factor in allograft inflammation and rejection. However, the role of cGAS/STING signaling-mediated innate immunity in the pathogenesis of allograft rejection remains unclear. Utilizing a well-established murine model of corneal transplantation, we demonstrated increased expression of cGAS and STING in rejected corneal allografts compared to syngeneic and normal corneas, along with significant activation of the cGAS/STING pathway, as evidenced by the enhanced phosphorylation of TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3). Pharmacological and genetic inhibition of cGAS/STING signaling markedly delayed corneal transplantation rejection, resulting in prolonged survival time and reduced inflammatory infiltration. Furthermore, we observed an increase in the formation of neutrophil extracellular traps (NETs) in rejected allografts, and the inhibition of NET formation through targeting peptidylarginine deiminase 4 (PADI4) and DNase I treatment significantly alleviated immune rejection and reduced cGAS/STING signaling activity. Conversely, subconjunctival injection of NETs accelerated corneal transplantation rejection and enhanced the activation of the cGAS/STING pathway. Collectively, these findings demonstrate that NETs contribute to the exacerbation of allograft rejection via cGAS/STING signaling, highlighting the targeting of the NETs/cGAS/STING signaling pathway as a potential strategy for prolonging allograft survival.

Identification of genetic suppressors for a BSCL2 lipodystrophy pathogenic variant in Caenorhabditis elegans.

Seipin (BSCL2), a conserved endoplasmic reticulum protein, plays a critical role in lipid droplet (LD) biogenesis and in regulating LD morphology, pathogenic variants of which are associated with Berardinelli-Seip congenital generalized lipodystrophy type 2 (BSCL2). To model BSCL2 disease, we generated an orthologous BSCL2 variant, seip-1(A185P), in Caenorhabditis elegans. In this study, we conducted an unbiased chemical mutagenesis screen to identify genetic suppressors that restore embryonic viability in the seip-1(A185P) mutant background. A total of five suppressor lines were isolated and recovered from the screen. The defective phenotypes of seip-1(A185P), including embryonic lethality and impaired eggshell formation, were significantly suppressed in each suppressor line. Two of the five suppressor lines also alleviated the enlarged LDs in the oocytes. We then mapped a suppressor candidate gene, lmbr-1, which is an ortholog of human limb development membrane protein 1 (LMBR1). The CRISPR/Cas9 edited lmbr-1 suppressor alleles, lmbr-1(S647F) and lmbr-1(P314L), both significantly suppressed embryonic lethality and defective eggshell formation in the seip-1(A185P) background. The newly identified suppressor lines offer valuable insights into potential genetic interactors and pathways that may regulate seipin in the lipodystrophy model.

A mutation in F-actin polymerization factor suppresses the distal arthrogryposis type 5 PIEZO2 pathogenic variant in Caenorhabditis elegans.

The mechanosensitive PIEZO channel family has been linked to over 26 disorders and diseases. Although progress has been made in understanding these channels at the structural and functional levels, the underlying mechanisms of PIEZO-associated diseases remain elusive. In this study, we engineered four PIEZO-based disease models using CRISPR/Cas9 gene editing. We performed an unbiased chemical mutagen-based genetic suppressor screen to identify putative suppressors of a conserved gain-of-function variant pezo-1[R2405P] that in human PIEZO2 causes distal arthrogryposis type 5 (DA5; p. R2718P). Electrophysiological analyses indicate that pezo-1(R2405P) is a gain-of-function allele. Using genomic mapping and whole-genome sequencing approaches, we identified a candidate suppressor allele in the C. elegans gene gex-3. This gene is an ortholog of human NCKAP1 (NCK-associated protein 1), a subunit of the Wiskott-Aldrich syndrome protein (WASP)-verprolin homologous protein (WAVE/SCAR) complex, which regulates F-actin polymerization. Depletion of gex-3 by RNAi, or with the suppressor allele gex-3(av259[L353F]), significantly increased brood size and ovulation rate, as well as alleviating the crushed oocyte phenotype of the pezo-1(R2405P) mutant. Expression of GEX-3 in the soma is required to rescue the brood size defects in pezo-1(R2405P) animals. Actin organization and orientation were disrupted and distorted in the pezo-1 mutants. Mutation of gex-3(L353F) partially alleviated these defects. The identification of gex-3 as a suppressor of the pathogenic variant pezo-1(R2405P) suggests that the PIEZO coordinates with the cytoskeleton regulator to maintain the F-actin network and provides insight into the molecular mechanisms of DA5 and other PIEZO-associated diseases.

The Vps13-like protein BLTP2 is pro-survival and regulates phosphatidylethanolamine levels in the plasma membrane to maintain its fluidity and function.

Lipid transport proteins (LTPs) facilitate nonvesicular lipid exchange between cellular compartments and have critical roles in lipid homeostasis1. A new family of bridge-like LTPs (BLTPs) is thought to form lipid-transporting conduits between organelles2. One, BLTP2, is conserved across species but its function is not known. Here, we show that BLTP2 and its homolog directly regulate plasma membrane (PM) fluidity by increasing the phosphatidylethanolamine (PE) level in the PM. BLTP2 localizes to endoplasmic reticulum (ER)-PM contact sites34, 5, suggesting it transports PE from the ER to the PM. We find BLTP2 works in parallel with another pathway that regulates intracellular PE distribution and PM fluidity6, 7. BLTP2 expression correlates with breast cancer aggressiveness8-10. We found BLTP2 facilitates growth of a human cancer cell line and sustains its aggressiveness in an in vivo model of metastasis, suggesting maintenance of PM fluidity by BLTP2 may be critical for tumorigenesis in humans.

Screening and Stability Evaluation of Freeze-Dried Protective Agents for a Live Recombinant Pseudorabies Virus Vaccine.

Infection of pigs with the pseudorabies virus (PRV) causes significant economic losses in the pig industry. Immunization with live vaccines is a crucial aspect in the prevention of pseudorabies in swine. The TK/gE/gI/11k/28k deleted pseudorabies vaccine is a promising alternative for the eradication of epidemic pseudorabies mutant strains. This study optimized the lyophilization of a heat-resistant PRV vaccine to enhance the quality of a live vaccine against the recombinant PRV rHN1201TK-/gE-/gI-/11k-/28k-. The A4 freeze-dried protective formulation against PRV was developed by comparing the reduction in virus titer after lyophilization and after seven days of storage at 37 °C. The formulation contains 1% gelatin, 5% trehalose, 0.5% poly-vinylpyrimidine (PVP), 0.5% thiourea, and 1% sorbitol. The A4 freeze-dried vaccine demonstrated superior protection and thermal stability. It experienced a freeze-dried loss of 0.31 Lg post-freeze-drying and a heat loss of 0.42 Lg after being stored at a temperature of 37 °C for 7 consecutive days. The A4 freeze-dried vaccine was characterized through XRD, FTIR, and SEM analyses, which showed that it possessed an amorphous structure with a consistent porous interior. The trehalose component of the vaccine formed stable hydrogen bonds with the virus. Long-term and accelerated stability studies were also conducted. The A4 vaccine maintained viral titer losses of less than 1.0 Lg when exposed to 25 °C for 90 days, 37 °C for 28 days, and 45 °C for 7 days. The A4 vaccine had a titer loss of 0.3 Lg after storage at 2-8 °C for 24 months, and a predicted shelf life of 6.61 years at 2-8 °C using the Arrhenius equation. The A4 freeze-dried vaccine elicited no side effects when used to immunize piglets and produced specific antibodies. This study provides theoretical references and technical support to improve the thermal stability of recombinant PRV rHN1201TK-/gE-/gI-/11k-/28k- vaccines.

Transmembrane protein 120A (TMEM-120A/TACAN) coordinates with PIEZO channel during Caenorhabditis elegans reproductive regulation.

Membrane protein TMEM120A (also known as TACAN) was presumed to be both a mechanically activated molecule and a lipid-modifying enzyme. TMEM120A has been identified as a negative regulator of the essential excitatory mechanosensitive protein PIEZO2. However, the extent to which TMEM120A mediates PIEZO2's activity during physiological processes remains largely unknown. In this study, we used the Caenorhabditis elegans reproductive tract to explore the functional contribution of tmem-120, the sole TMEM120A/B ortholog, and its genetic interaction with pezo-1 in vivo. tmem-120 was expressed throughout the C. elegans development, particularly in the germline, embryos, and spermatheca. A tmem-120 mutant with a full-length deletion (tmem-120Δ) displayed deformed germline, maternal sterility, and a reduced brood size. In vivo live imaging revealed that pinched zygotes were frequently observed in the uterus of tmem-120Δ mutant animals, suggesting damage during spermathecal contraction. We then employed the auxin-inducible degradation system to degrade TMEM-120 protein in all somatic tissues or the germline, both of which resulted in reduced brood sizes. These findings suggested that multiple inputs of tmem-120 from different tissues regulate reproduction. Lastly, the loss of tmem-120 alleviated the brood size reduction and defective sperm navigation behavior in the pezo-1Δ mutant. Overall, our findings reveal a role for tmem-120 in regulating reproductive physiology in C. elegans, and suggest an epistatic interaction between pezo-1 and tmem-120 when governing proper reproduction.

Andy Golden: Mentorship through the Years.

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Identification of Genetic Suppressors for a Berardinelli-Seip Congenital Generalized Lipodystrophy Type 2 (BSCL2) Pathogenic Variant in C. elegans.

Maintaining the metabolic homeostasis of fatty acids is crucial for human health. Excess fatty acids are stored in lipid droplets (LDs), the primary energy reservoir that helps regulate fat and lipid homeostasis in nearly all cell types. Seipin (BSCL2), a conserved endoplasmic reticulum protein, plays a critical role in LD biogenesis and regulating LD morphology. Pathogenic variants of seipin are associated with multiple human genetic diseases, including Berardinelli-Seip Congenital Generalized Lipodystrophy Type 2 (BSCL2). However, the cellular and molecular mechanisms by which dysfunctional seipin leads to these diseases remain unclear. To model BSCL2 disease, we generated an orthologous BSCL2 pathogenic variant seip-1(A185P) using CRISPR/Cas9 genome editing in Caenorhabditis elegans . This variant led to severe developmental and cellular defects, including embryonic lethality, impaired eggshell formation, and abnormally enlarged LDs. We set out to identify genetic determinants that could suppress these defective phenotypes in the seip-1(A185P) mutant background. To this end, we conducted an unbiased chemical mutagenesis screen to identify genetic suppressors that restore embryonic viability in the seip-1(A185P) mutant background. A total of five suppressor lines were isolated and recovered from the screen. The defective phenotypes of seip-1(A185P) , including embryonic lethality and impaired eggshell formation, were significantly suppressed in each suppressor line. Two of the five suppressor lines also alleviated the enlarged LDs in the oocytes. We then mapped a suppressor candidate gene, R05D3.2 (renamed as lmbr-1 ), which is an ortholog of human LMBR1 (limb development membrane protein 1). The CRISPR/Cas9 edited lmbr-1 suppressor alleles, lmbr-1(Ser647Phe) and lmbr-1(Pro314Leu) , both significantly suppressed embryonic lethality and defective eggshell formation in the seip-1(A185P) background. The newly identified suppressor lines offer valuable insights into potential genetic interactors and pathways that may regulate seipin in the lipodystrophy model.

Using CRISPR knock-in of fluorescent tags to examine isoform-specific expression of EGL-19 in C. elegans.

L-type voltage-gated calcium channels (VGCCs) regulate calcium influx and excitation-contraction coupling in many types of muscle cells. Thus, VGCC mutations can cause skeletal and cardiac muscle diseases in humans, such as Duchenne muscular dystrophy and Timothy syndrome. To better understand the genetics and native expression of VGCCs, we have chosen to use the microscopic roundworm, C. elegans . The egl-19 locus is the sole L-type VGCC gene and it encodes three distinct isoforms (a, b, and c). Isoform c is curious because the protein is truncated, lacking the transmembrane domains that form the physical calcium channel. In this study, we have characterized egl-19 expression using CRISPR/Cas9 genome editing to 'knock-in' fluorescent tags of differing colors, allowing us to distinguish the expression pattern of each isoform. Not surprisingly, we found that EGL-19 is expressed in all types of muscle. In addition, we provide evidence that the truncated c isoform is expressed. Finally, although we find evidence that specific isoforms can have unique subcellular distributions, we also observed some expression patterns that appear to be artifacts. Overall, our results show interesting patterns of egl-19 expression, but also highlight the need for caution when interpreting expression of reporter genes even when they represent endogenous tags.

Mutation in F-actin Polymerization Factor Suppresses Distal Arthrogryposis Type 5 (DA5) PIEZO2 Pathogenic Variant in Caenorhabditis elegans.

The mechanosensitive PIEZO channel family has been linked to over 26 disorders and diseases. Although progress has been made in understanding these channels at the structural and functional levels, the underlying mechanisms of PIEZO-associated diseases remain elusive. In this study, we engineered four PIEZO-based disease models using CRISPR/Cas9 gene editing. We performed an unbiased chemical mutagen-based genetic suppressor screen to identify putative suppressors of a conserved gain-of-function variant pezo-1[R2405P] that in human PIEZO2 causes distal arthrogryposis type 5 (DA5; p. R2718P). Electrophysiological analyses indicate that pezo-1(R2405P) is a gain-of-function allele. Using genomic mapping and whole genome sequencing approaches, we identified a candidate suppressor allele in the C. elegans gene gex-3. This gene is an ortholog of human NCKAP1 (NCK-associated protein 1), a subunit of the Wiskott-Aldrich syndrome protein (WASP)-verprolin homologous protein (WAVE/SCAR) complex, which regulates F-actin polymerization. Depletion of gex-3 by RNAi, or with the suppressor allele gex-3(av259[L353F]) , significantly restored the small brood size and low ovulation rate, as well as alleviated the crushed oocyte phenotype of the pezo-1(R2405P) mutant. Auxin-inducible degradation of GEX-3 revealed that only somatic-specific degradation of GEX-3 restored the reduced brood size in the pezo-1(R2405P) mutants. Additionally, actin organization and orientation were disrupted and distorted in the pezo-1 mutants. Mutation of gex-3(L353F) partially alleviated these defects. The identification of gex-3 as a suppressor of the pathogenic variant pezo-1(R2405P) suggests that the cytoskeleton plays an important role in regulating PIEZO channel activity and provides insight into the molecular mechanisms of DA5 and other PIEZO-associated diseases.

A missense mutation in the C. elegans src-2 tyrosine-protein kinase reduces brood size and enhances embryonic morphogenesis defects in src-1(RNAi) conditions.

Goldenhar Syndrome is a rare congenital disorder characterized by hemifacial microsomia. Although select mutations have been mapped for this disorder, the genetic etiologies in the majority of cases remain unknown. A recent clinical report of a Goldenhar Syndrome patient identified a homozygous missense mutation in FRK , a gene associated with various types of cancer. In this work, we precisely modeled the disease-associated missense mutation in the C. elegans FRK ortholog src-2 , using CRISPR/Cas9 gene editing, and investigated the physiological role of this mutation and the src-2 gene. In addition, we generated a conserved variant in src-1 ( FYN ortholog) to assess the functional redundancy of the conserved variant. The putative pathogenic variants src-1 (Val190Ile) or src-2 (Val170Ile) caused only subtle phenotypes, suggesting that these mutations alone are not sufficient to explain the facial deformities observed in the Goldenhar Syndrome patient. However, the src-2 (Val170Ile) mutant exhibited reduced brood size and moderately enhanced embryonic developmental phenotypes, including epidermal and neuronal patterning defects, in the src-1 (RNAi) condition, indicating that the src-2 (Val170Ile) locus could play a supportive role during developmental processes. Overall, however, these studies showed that src-1 /FYN is essential for regulating embryogenesis and morphogenesis, while src-2 /FRK is largely dispensable for normal embryonic development, suggesting FYN , not FRK , is the dominant non-receptor protein kinase during embryonic development in C. elegans .

Pathogenicity characterization of PRRSV-1 181187-2 isolated in China.

PRRSV-1 has caused more clinical infections in pigs in Chinese swine herds in recent years, however, the pathogenicity of PRRSV-1 in China is unclear. In order to study the pathogenicity of PRRSV-1, in this study, a PRRSV-1 strain, 181187-2, was isolated in primary alveolar macrophage (PAM) cells from a farm where abortions had been reported in China. The complete genome of 181187-2 was 14932 bp excluding Poly A, with 54-amino acid continuous deletion in the Nsp2 gene and 1 amino deletion in ORF3 gene compared with LV. Additionally, the piglets inoculated with strain 181187-2 by intranasal and intranasal plus intramuscular injection, animal experiments showed clinical symptoms including transient fever and depression, with no death. The obvious histopathological lesions including interstitial pneumonia and lymph node hemorrhage, and there were no significant differences in clinical symptoms and histopathological lesions with different challenge ways. Our results indicated that PRRSV -1 181187-2 was a moderately pathogenic strain in piglets.

Hyperglycemia Induces Tear Reduction and Dry Eye in Diabetic Mice through the Norepinephrine-α1 Adrenergic Receptor-Mitochondrial Impairment Axis of Lacrimal Gland.

Dry eye syndrome is a common complication in diabetic patients with a prevalence of up to 54.3%. However, the pathogenic mechanisms underlying hyperglycemia-induced tear reduction and dry eye remain less understood. The present study indicated that both norepinephrine (NE) and tyrosine hydroxylase levels were elevated in the lacrimal gland of diabetic mice, accompanied by increased Fos proto-oncogene (c-FOS)+ cells in the superior cervical ganglion. However, the elimination of NE accumulation by surgical and chemical sympathectomy significantly ameliorated the reduction in tear production, suppressed abnormal inflammation of the lacrimal gland, and improved the severity of dry eye symptoms in diabetic mice. Among various adrenergic receptors (ARs), the α1 subtype played a predominant role in the regulation of tear production, as treatments of α1AR antagonists improved tear secretion in diabetic mice compared with ßAR antagonist propranolol. Moreover, the α1AR antagonist alfuzosin treatment also alleviated functional impairments of the meibomian gland and goblet cells in diabetic mice. Mechanically, the α1AR antagonist rescued the mitochondrial bioenergetic deficit, increased the mitochondrial DNA copy numbers, and elevated the glutathione levels of the diabetic lacrimal gland. Overall, these results deciphered a previously unrecognized involvement of the NE-α1AR-mitochondrial bioenergetics axis in the regulation of tear production in the lacrimal gland, which may provide a potential strategy to counteract diabetic dry eye by interfering with the α1AR activity.

Paracondylar process combined with persistent first intersegmental vertebral artery: an anatomic case report and literature review.

The paracondylar process (PCP) and the persistent first intersegmental vertebral artery (PFIA) are both rare variations at the craniovertebral junction. We report the above two variations coexisting in one cadaveric head during the training of far lateral approach in our skull base lab. The specimen simultaneously had a left occipitalized atlas associated with a PFIA and a right PCP. The previous reports, the embryogenesis, and the clinical implications of the two variations were also reviewed. Preoperative recognition of the rare variations is essential to a safe far lateral approach.

The mounted alloimmunity of the iris-ciliary body devotes a hotbed of immune cells for corneal transplantation rejection.

Graft rejection is still the major obstacle causing corneal transplantation failure. However, the underlying pathogenesis remains largely unclear. The iris-ciliary body (I-C) is enriched with blood vessels and various immune cell populations, presumably predisposed to be involved in corneal transplantation rejection. After penetrating keratoplasty, compared to the normal (Nor) and syngeneic (Syn) groups, I-C tissues in the allogeneic (Allo) group displayed stronger alloimmune responses, with more infiltrations of CD45+ inflammatory cells and CD3+ lymphocytes, increased transcriptional levels of pro-inflammatory cytokines, and elevated NF-κB activity. This histopathology was similar to the pathological alterations of corneal allografts. Angiography analysis revealed the abnormal vasculature in the iris during allograft rejection, characterized by vasodilatation, increased vessel density, and vascular permeability. While, immunofluorescence staining showed the intact tight junction of the posterior iris epithelium. In vitro, human microvascular endothelial cells (HMECs) stimulated by tumor necrosis factor-α (TNF-α) showed an increased Evans blue (EB)-albumin leakage, with lower expression of zonula occludens-1 (ZO-1) and Occludin. The increased EB-albumin leakage, up-regulated NF-κB activity, and reduced expression of ZO-1 and Occludin could be partially reversed after cyclosporine A (CsA) administration. In contrast, the barrier function in primary mouse iris pigment epithelial cells (IPEs) after TNF-α treatment remained largely unchanged. These findings revealed the vigorous alloimmunity in I-C tissues, characterized with impaired vascularization but intact posterior epithelial barrier in the iris, which allowed proteins and immune cells to be exudated from the front surface of I-C tissues, and facilitated immune reaction in the anterior chamber, thereby contributing to aggravated corneal transplantation rejection.

lncNBAT1/APOBEC3A is a mediator of HBX-induced chemoresistance in diffuse large B cell lymphoma cells.

Individuals with diffuse large B cell lymphoma (DLBCL) infected with hepatitis B virus (HBV) have worse chemotherapy efficacy and poorer outcomes. It is still unclear whether long noncoding RNAs (lncRNAs) serve as prognostic and therapeutic targets in the chemotherapy resistance of individuals with DLBCL and HBV infection. Here we found that the core component of HBV (HBX) directly upregulated the expression of lncNBAT1, which was closely associated with the chemotherapy outcomes of HBV-infected individuals with DLBCL. Upregulation of lncNBAT1 reduced the sensitivity of DLBCL cells to chemotherapeutic agents (methotrexate [MTX] or cytarabine [Ara-C]) that induced S phase arrest, whereas knockdown of lncNBAT1 significantly relieved the chemoresistance of HBX-expressing DLBCLs. Mechanistically, lncNBAT1 could interact with the signal transducer and activator of transcription 1 (STAT1) to prevent its enrichment at the promoter region of the functional target gene apolipoprotein B mRNA editing enzyme catalytic subunit 3A (APOBEC3A), inhibiting expression of APOBEC3A and inducing resistance to MTX in DLBCL cells. Furthermore, clinical data analysis showed that lncNBAT1 and APOBEC3A expression was closely related to the poor prognosis and short survival of individuals with DLBCL. Our findings suggest a potential prognostic marker and a candidate lncRNA target for treating HBV-infected individuals with DLBCL.

Vps13-like proteins provide phosphatidylethanolamine for GPI anchor synthesis in the ER.

Glycosylphosphatidylinositol (GPI) is a glycolipid membrane anchor found on surface proteins in all eukaryotes. It is synthesized in the ER membrane. Each GPI anchor requires three molecules of ethanolamine phosphate (P-Etn), which are derived from phosphatidylethanolamine (PE). We found that efficient GPI anchor synthesis in Saccharomyces cerevisiae requires Csf1; cells lacking Csf1 accumulate GPI precursors lacking P-Etn. Structure predictions suggest Csf1 is a tube-forming lipid transport protein like Vps13. Csf1 is found at contact sites between the ER and other organelles. It interacts with the ER protein Mcd4, an enzyme that adds P-Etn to nascent GPI anchors, suggesting Csf1 channels PE to Mcd4 in the ER at contact sites to support GPI anchor biosynthesis. CSF1 has orthologues in Caenorhabditis elegans (lpd-3) and humans (KIAA1109/TWEEK); mutations in KIAA1109 cause the autosomal recessive neurodevelopmental disorder Alkuraya-Kucinskas syndrome. Knockout of lpd-3 and knockdown of KIAA1109 reduced GPI-anchored proteins on the surface of cells, suggesting Csf1 orthologues in human cells support GPI anchor biosynthesis.

Distinct mechanoreceptor pezo-1 isoforms modulate food intake in the nematode Caenorhabditis elegans.

Two PIEZO mechanosensitive cation channels, PIEZO1 and PIEZO2, have been identified in mammals, where they are involved in numerous sensory processes. While structurally similar, PIEZO channels are expressed in distinct tissues and exhibit unique properties. How different PIEZOs transduce force, how their transduction mechanism varies, and how their unique properties match the functional needs of the tissues they are expressed in remain all-important unanswered questions. The nematode Caenorhabditis elegans has a single PIEZO ortholog (pezo-1) predicted to have 12 isoforms. These isoforms share many transmembrane domains but differ in those that distinguish PIEZO1 and PIEZO2 in mammals. We used transcriptional and translational reporters to show that putative promoter sequences immediately upstream of the start codon of long pezo-1 isoforms predominantly drive green fluorescent protein (GFP) expression in mesodermally derived tissues (such as muscle and glands). In contrast, sequences upstream of shorter pezo-1 isoforms resulted in GFP expression primarily in neurons. Putative promoters upstream of different isoforms drove GFP expression in different cells of the same organs of the digestive system. The observed unique pattern of complementary expression suggests that different isoforms could possess distinct functions within these organs. We used mutant analysis to show that pharyngeal muscles and glands require long pezo-1 isoforms to respond appropriately to the presence of food. The number of pezo-1 isoforms in C. elegans, their putative differential pattern of expression, and roles in experimentally tractable processes make this an attractive system to investigate the molecular basis for functional differences between members of the PIEZO family of mechanoreceptors.