Integrating transcriptomics, glycomics and glycoproteomics to characterize hepatitis B virus-associated hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) ranks as the third most common cause of cancer related death globally, representing a substantial challenge to global healthcare systems. In China, the primary risk factor for HCC is the hepatitis B virus (HBV). Aberrant serum glycoconjugate levels have long been linked to the progression of HBV-associated HCC (HBV-HCC). Nevertheless, few study systematically explored the dysregulation of glycoconjugates in the progression of HBV-associated HCC and their potency as the diagnostic and prognostic biomarker. METHODS: An integrated strategy that combined transcriptomics, glycomics, and glycoproteomics was employed to comprehensively investigate the dynamic alterations in glyco-genes, N-glycans, and glycoproteins in the progression of HBV- HCC. RESULTS: Bioinformatic analysis of Gene Expression Omnibus (GEO) datasets uncovered dysregulation of fucosyltransferases (FUTs) in liver tissues from HCC patients compared to adjacent tissues. Glycomic analysis indicated an elevated level of fucosylated N-glycans, especially a progressive increase in fucosylation levels on IgA1 and IgG2 determined by glycoproteomic analysis. CONCLUSIONS: The findings indicate that the abnormal fucosylation plays a pivotal role in the progression of HBV-HCC. Systematic and integrative multi-omic analysis is anticipated to facilitate the discovery of aberrant glycoconjugates in tumor progression.

Molecular Detection and Genetic Diversity of Cytomegaloviruses and Lymphocryptoviruses in Free-Roaming and Captive African Green Monkeys (Chlorocebus sabaeus).

To date, limited information is available on cytomegalovirus (CMV) and lymphocryptovirus (LCV) from Chlorocebus monkeys. We report here high detection rates of herpesviruses in free-roaming African green monkeys (AGMs, Chlorocebus sabaeus) (26.4%, 23/87) and in captive AGMs (75%, 3/4) with respiratory disease on the Caribbean Island of St. Kitts. LCV (81.25%) was more prevalent than CMV (18.75%) in the AGMs. Applying a bigenic PCR approach (targeting DNA polymerase (DPOL) and glycoprotein B (gB) genes), long sequences were obtained from representative AGM CMV (KNA-SD6) and LCV (KNA-E4, -N6 and -R15) samples, and mixed LCV infections were identified in KNA-N6 and -R15. The nucleotide (nt) sequence (partial DPOL-intergenic region-partial gB) and partial DPOL- and gB-amino acid (aa) sequences of AGM CMV KNA-SD6 were closely related to Cytomegalovirus cercopithecinebeta5 isolates from grivet monkeys, whilst those of AGM LCV KNA-E4 and -N6 (and E4-like gB of KNA-R15) were more closely related to cognate sequences of erythrocebus patas LCV1 from patas monkey than other LCVs, corroborating the concept of cospeciation in the evolution of CMV/LCV. On the other hand, the partial DPOL aa sequence of KNA-R15, and additional gB sequences (N6-gB-2 and R15-gB-2) from samples KNA-N6 and -R15 (respectively) appeared to be distinct from those of Old World monkey LCVs, indicating LCV evolutionary patterns that were not synchronous with those of host species. The present study is the first to report the molecular prevalence and genetic diversity of CMV/LCV from free-roaming/wild and captive AGMs, and is the first report on analysis of CMV nt/deduced aa sequences from AGMs and LCV gB sequences from Chlorocebus monkeys.

Restoration of virulence in the attenuated Candid#1 vaccine virus requires reversion at both positions 168 and 427 in the envelope glycoprotein GPC.

Live-attenuated virus vaccines provide long-lived protection against viral disease but carry inherent risks of residual pathogenicity and genetic reversion. The live-attenuated Candid#1 vaccine was developed to protect Argentines against lethal infection by the Argentine hemorrhagic fever arenavirus, Junín virus. Despite its safety and efficacy in Phase III clinical study, the vaccine is not licensed in the US, in part due to concerns regarding the genetic stability of attenuation. Previous studies had identified a single F427I mutation in the transmembrane domain of the Candid#1 envelope glycoprotein GPC as the key determinant of attenuation, as well as the propensity of this mutation to revert upon passage in cell culture and neonatal mice. To ascertain the consequences of this reversion event, we introduced the I427F mutation into recombinant Candid#1 (I427F rCan) and investigated the effects in two validated small-animal models: in mice expressing the essential virus receptor (human transferrin receptor 1; huTfR1) and in the conventional guinea pig model. We report that I427F rCan displays only modest virulence in huTfR1 mice and appears attenuated in guinea pigs. Reversion at another attenuating locus in Candid#1 GPC (T168A) was also examined, and a similar pattern was observed. By contrast, virus bearing both revertant mutations (A168T+I427F rCan) approached the lethal virulence of the pathogenic Romero strain in huTfR1 mice. Virulence was less extreme in guinea pigs. Our findings suggest that genetic stabilization at both positions is required to minimize the likelihood of reversion to virulence in a second-generation Candid#1 vaccine.IMPORTANCELive-attenuated virus vaccines, such as measles/mumps/rubella and oral poliovirus, provide robust protection against disease but carry with them the risk of genetic reversion to the virulent form. Here, we analyze the genetics of reversion in the live-attenuated Candid#1 vaccine that is used to protect against Argentine hemorrhagic fever, an often-lethal disease caused by the Junín arenavirus. In two validated small-animal models, we find that restoration of virulence in recombinant Candid#1 viruses requires back-mutation at two positions specific to the Candid#1 envelope glycoprotein GPC, at positions 168 and 427. Viruses bearing only a single change showed only modest virulence. We discuss strategies to genetically harden Candid#1 GPC against these two reversion events in order to develop a safer second-generation Candid#1 vaccine virus.

Somatic Mutations within Myocilin due to Aging May Be a Potential Risk Factor for Glaucoma.

Glaucoma is a chronic optic neuropathy that leads to irreversible vision loss. Aging and family history are the two most important risk factors of glaucoma. One of the most studied genes involved in the onset of open-angle glaucoma is myocilin (MYOC). About 105 germline mutations within MYOC are known to be associated with glaucoma and result in endoplasmic reticulum (ER) stress, which leads to trabecular meshwork (TM) cell death and subsequent intraocular pressure (IOP) elevation. However, only about 4% of the population carry these mutations. An analysis of MYOC somatic cancer-associated mutations revealed a notable overlap with pathogenic glaucoma variants. Because TM cells have the potential to accumulate somatic mutations at a rapid rate due to ultraviolet (UV) light exposure, we propose that an accumulation of somatic mutations within MYOC is an important contributor to the onset of glaucoma.

Self-assembling ferritin nanoplatform for the development of infectious hematopoietic necrosis virus vaccine.

Self-assembling protein nanoparticles are used as a novel vaccine design platform to improve the stability and immunogenicity of safe subunit vaccines, while providing broader protection against viral infections. Infectious Hematopoietic Necrosis virus (IHNV) is the causative agent of the WOAH-listed IHN diseases for which there are currently no therapeutic treatments and no globally available commercial vaccine. In this study, by genetically fusing the virus glycoprotein to the H. pylori ferritin as a scaffold, we constructed a self-assembling IHNV nanovaccine (FerritVac). Despite the introduction of an exogenous fragment, the FerritVac NPs show excellent stability same as Ferritin NPs under different storage, pH, and temperature conditions, mimicking the harsh gastrointestinal condition of the virus main host (trout). MTT viability assays showed no cytotoxicity of FerritVac or Ferritin NPs in zebrafish cell culture (ZFL cells) incubated with different doses of up to 100 µg/mL for 14 hours. FerritVac NPs also upregulated expression of innate antiviral immunity, IHNV, and other fish rhabdovirus infection gene markers (mx, vig1, ifit5, and isg-15) in the macrophage cells of the host. In this study, we demonstrate the development of a soluble recombinant glycoprotein of IHNV in the E. coli system using the ferritin self-assembling nanoplatform, as a biocompatible, stable, and effective foundation to rescue and produce soluble protein and enable oral administration and antiviral induction for development of a complete IHNV vaccine. This self-assembling protein nanocages as novel vaccine approach offers significant commercial potential for non-mammalian and enveloped viruses.

Application of CRISPR/Cas9 for Rapid Genome Editing of Pseudorabies Virus and Bovine Herpesvirus-1.

The CRISPR/Cas9 system is widely used to manipulate viral genomes. Although Alphaherpesvirinae genomes are large and complicated to edit, in recent years several Pseudorabies virus (PRV) mutants have been successfully generated using the CRISPR/Cas9 system. However, the application of CRISPR/Cas9 editing on another member of alpha herpesviruses, bovine herpesvirus-1 (BHV-1), is rarely reported. This paper reports a rapid and straightforward approach to manipulating herpesviruses genome using CRISPR/Cas9. The recombinant plasmids contained the left and right arm of the thymidine kinase (TK) gene of PRV or of the glycoprotein I (gI) and glycoprotein E (gE) of BHV-1. Upon the cleavage of the TK or gIgE gene by Cas9 protein, this was replaced by the enhanced green fluorescence protein (eGFP) by homologous recombination. With this approach, we generated recombinant TK-/eGFP+ PRV and gIgE-/eGFP+ BHV-1 mutants and then proceeded to characterize their biological activities in vitro and in vivo. In conclusion, we showed that alpha herpesvirus, including PRV and BHV-1, can be rapidly edited using the CRISPR/Cas9 approach paving the way to the development of animal herpesvirus vaccines.

LIGHT regulated gene expression in rheumatoid synovial fibroblasts.

BACKGROUND: Synovial hyperplasia caused by rheumatoid arthritis (RA), an autoimmune inflammatory disease, leads to the destruction of the articular cartilage and bone. A member of the tumor necrosis factor superfamily, Lymphotoxin-related inducible ligand that competes for glycoprotein D binding to herpes virus entry mediator on T cells (LIGHT) has been shown to correlate with the pathogenesis of RA. METHODS: We used cDNA microarray analysis to compare the expression of genes in rheumatoid fibroblast-like synoviocytes with and without LIGHT stimulation. RESULTS: Significant changes in gene expression (P-values < 0.05 and fold change ≥ 2.0) were associated mainly with biological function categories of glycoprotein, glycosylation site as N-linked, plasma membrane part, integral to plasma membrane, intrinsic to plasma membrane, signal, plasma membrane, signal peptide, alternative splicing, and topological domain as extracellular. CONCLUSIONS: Our results indicate that LIGHT may regulate the expression in RA-FLS of genes which are important in the differentiation of several cell types and in cellular functions.

Herpes Simplex Virus 1 Glycoprotein B from a Hyperfusogenic Virus Mediates Enhanced Cell-Cell Fusion.

Herpes simplex virus 1 (HSV-1) causes significant morbidity and death in humans worldwide. Herpes simplex virus 1 has a complex fusion mechanism that is incompletely understood. The HSV-1 strain ANG has notable fusion and entry activities that distinguish it from wild type. HSV-1 ANG virions fused with the Vero cell surface at 4 °C and also entered cells more efficiently at 15 °C, relative to wild type HSV-1 strain KOS virions, consistent with a hyperfusogenic phenotype. Understanding the molecular basis for the unique entry and fusion activities of HSV-1 strain ANG will help decipher the HSV fusion reaction and entry process. Sequencing of HSV-1 ANG genes revealed multiple changes in gB, gC, gD, gH, and gL proteins relative to wild type HSV-1 strains. The ANG UL45 gene sequence, which codes for a non-essential envelope protein, was identical to wild type KOS. HSV-1 ANG gB, gD, and gH/gL were necessary and sufficient to mediate cell-cell fusion in a virus-free reporter assay. ANG gB, when expressed with wild type KOS gD and gH/gL, increased membrane fusion, suggesting that ANG gB has hyperfusogenic cell-cell fusion activity. Replacing the KOS gD, gH, or gL with the corresponding ANG alleles did not enhance cell-cell fusion. The novel mutations in the ANG fusion and entry glycoproteins provide a platform for dissecting the cascade of interactions that culminate in HSV fusion and entry.

[Is it possible to detect surface antigen CD133 on patient-derived glioblastoma continuous cell cultures using fluorescent aptamers?] / Vozmozhna li detektsiya poverkhnostnogo antigena CD133 na perevivaemykh kul'turakh kletok glioblastomy patsientov s pomoshch'yu fluorestsentnykh aptamerov?

Theranostics combines diagnostics and therapeutic exposure. Regarding glioblastomas, theranostics solves the problem of detecting and destroying tumor stem cells resistant to irradiation and chemotherapy and causing tumor recurrence. Transmembrane surface antigen CD133 is considered as a potential marker of tumor stem cells. OBJECTIVE: To detect CD133 in patient-derived glioblastoma continuous cell cultures using fluorescence microscopy and modified aptamers (molecular recognition elements) anti-CD133. MATERIAL AND METHODS: To detect CD133, we used mousey fluorescence monoclonal antibodies anti-CD133 MA1-219, FAM-modified DNA aptamers anti-CD133 AP-1-M and Cs5. Non-aptamer DNA oligonucleotide NADO was used as a negative control. Detection was performed for three samples of patient-derived glioblastoma continuous cell cultures coded as 1548, 1721 and 1793. RESULTS: MA1-219 antibodies brightly stained cell culture 1548, to a lesser extent - 1721. There was diffuse staining of cell culture 1793. Cs5-FAM aptamer stained cells in a similar way, but much weaker. AP-1-M-FAM aptamer interacted with cells even weaker and diffusely stained only cell culture 1793. Non-aptamer NADO did not stain cell culture 1548 and very weakly diffusely stained cell culture 1793. CONCLUSION: For both molecular recognition elements (MA1-219 antibody and Cs5 aptamer), 3 cell culture samples can be arranged in the following order possibly reflecting CD133 status decrease: strong signal for cell culture 1548, much weaker for 1721, even weaker for 1793. Only cell culture 1548 can be considered CD133 positive with combination of Cs5+ and NADO signals. Cell culture 1793 is CD133 false positive with combination of Cs5+ and NADO+ signals.

Effects of autophagy-inhibiting chemicals on sialylation of Fc-fusion glycoprotein in recombinant CHO cells.

The occurrence of autophagy in recombinant Chinese hamster ovary (rCHO) cell culture has attracted attention due to its effects on therapeutic protein production. Given the significance of glycosylation in therapeutic proteins, this study examined the effects of autophagy-inhibiting chemicals on sialylation of Fc-fusion glycoproteins in rCHO cells. Three chemical autophagy inhibitors known to inhibit different stages were separately treated with two rCHO cell lines that produce the same Fc-fusion glycoprotein derived from DUKX-B11 and DG44. All autophagy inhibitors significantly decreased the sialylation of Fc-fusion glycoprotein in both cell lines. The decrease in sialylation of Fc-fusion glycoprotein is unlikely to be attributed to the release of intracellular enzymes, given the high cell viability and low activity of extracellular sialidases. Interestingly, the five intracellular nucleotide sugars remained abundant in cells treated with autophagy inhibitors. In the mRNA expression profiles of 27 N-glycosylation-related genes using the NanoString nCounter system, no significant differences in gene expression were noted. With the positive effect of supplementing nucleotide sugar precursors on sialylation, attempts were made to enhance the levels of intracellular nucleotide sugars by supplying these precursors. The addition of nucleotide sugar precursors to cultures treated with inhibitors successfully enhanced the sialylation of Fc-fusion glycoproteins compared to the control culture. This was particularly evident under mild stress conditions and not under relatively severe stress conditions, which were characterized by a high decrease in sialylation. These results suggest that inhibiting autophagy in rCHO cell culture decreases sialylation of Fc-fusion glycoprotein by constraining the availability of intracellular nucleotide sugars. KEY POINTS: •  The autophagy inhibition in rCHO cell culture leads to a significant reduction in the sialylation of Fc-fusion glycoprotein. •  The pool of five intracellular nucleotide sugars remained highly abundant in cells treated with autophagy inhibitors. •  Supplementation of nucleotide sugar precursors effectively restores decreased sialylation, particularly under mild stress conditions but not in relatively severe stress conditions.

Production of Influenza Virus Glycoproteins Using Insect Cells.

The baculovirus/insect cell expression system is a very useful tool for reagent and antigen generation in vaccinology, virology, and immunology. It allows for the production of recombinant glycoproteins, which are used as antigens in vaccination studies and as reagents in immunological assays. Here, we describe the process of recombinant glycoprotein production using the baculovirus/insect cell expression system.

A single-point mutation in the rubella virus E1 glycoprotein promotes rescue of recombinant vesicular stomatitis virus.

Rubella virus (RuV) is an enveloped plus-sense RNA virus and a member of the Rubivirus genus. RuV infection in pregnant women can lead to miscarriage or an array of severe birth defects known as congenital rubella syndrome. Novel rubiviruses were recently discovered in various mammals, highlighting the spillover potential of other rubiviruses to humans. Many features of the rubivirus infection cycle remain unexplored. To promote the study of rubivirus biology, here, we generated replication-competent recombinant VSV-RuV (rVSV-RuV) encoding the RuV transmembrane glycoproteins E2 and E1. Sequencing of rVSV-RuV showed that the RuV glycoproteins acquired a single-point mutation W448R in the E1 transmembrane domain. The E1 W448R mutation did not detectably alter the intracellular expression, processing, glycosylation, colocalization, or dimerization of the E2 and E1 glycoproteins. Nonetheless, the mutation enhanced the incorporation of RuV E2/E1 into VSV particles, which bud from the plasma membrane rather than the RuV budding site in the Golgi. Neutralization by E1 antibodies, calcium dependence, and cell tropism were comparable between WT-RuV and either rVSV-RuV or RuV containing the E1 W448R mutation. However, the E1 W448R mutation strongly shifted the threshold for the acid pH-triggered virus fusion reaction, from pH 6.2 for the WT RuV to pH 5.5 for the mutant. These results suggest that the increased resistance of the mutant RuV E1 to acidic pH promotes the ability of viral envelope proteins to generate infectious rVSV and provide insights into the regulation of RuV fusion during virus entry and exit.IMPORTANCERubella virus (RuV) infection in pregnant women can cause miscarriage or severe fetal birth defects. While a highly effective vaccine has been developed, RuV cases are still a significant problem in areas with inadequate vaccine coverage. In addition, related viruses have recently been discovered in mammals, such as bats and mice, leading to concerns about potential virus spillover to humans. To facilitate studies of RuV biology, here, we generated and characterized a replication-competent vesicular stomatitis virus encoding the RuV glycoproteins (rVSV-RuV). Sequence analysis of rVSV-RuV identified a single-point mutation in the transmembrane region of the E1 glycoprotein. While the overall properties of rVSV-RuV are similar to those of WT-RuV, the mutation caused a marked shift in the pH dependence of virus membrane fusion. Together, our studies of rVSV-RuV and the identified W448R mutation expand our understanding of rubivirus biology and provide new tools for its study.

Lrg1 silencing attenuates ischemia-reperfusion renal injury by regulating autophagy and apoptosis through the TGFß1- Smad1/5 signaling pathway.

BACKGROUND: Dysfunction in the processes of autophagy and apoptosis within renal tubular epithelial cells (RTEc) contributes to renal ischemia-reperfusion injury (IRI). However, the factors influencing this dysfunction remain unclear. Leucine-rich alpha-2-glycoprotein 1 (Lrg1) plays a role in the progression of diabetic nephropathy and kidney fibrosis by modulating the activin receptor-like kinase 1 (ALK1)-Smad1/5/8 and TGF-ß1/Smad3 pathways, respectively. Therefore, we aimed to investigate whether Lrg1 is involved in the pathological mechanisms of renal IRI and whether its effects are related to the dysregulation of autophagy and apoptosis in RTEc. METHODS: We conducted in vitro and in vivo experiments using CoCl2-induced hypoxic human kidney-2 (HK-2) cells and mice with renal IRI, respectively. Lrg1 was silenced using siRNA and lentiviral vectors in HK-2 cells and mouse kidneys. Rapamycin (Rapa) and methyladenine were applied to regulate autophagy in renal IRI models. RESULTS: Increased Lrg1 expression was observed in hypoxic HK-2 cells and in the kidneys of mice with renal IRI. Silencing of Lrg1 through siRNA and lentiviral approaches restored autophagy and suppressed apoptosis in CoCl2-induced hypoxic HK-2 cells and renal IRI models. Additionally, reduced Lrg1 expression alleviated kidney damage caused by renal IRI. The downregulation of Lrg1 expression restrained the TGFß-Smad1/5 signaling pathway in hypoxic-induced HK-2 cells and renal IRI by reducing ALK1 expression. Lastly, the enhancement of autophagy, achieved through Rapa treatment, provided protection against renal IRI in mice. CONCLUSIONS: Our findings suggest that Lrg1 silencing can be applied as a potential therapeutic target to inhibit the TGFß1-Smad1/5 pathway, thereby enhancing autophagy and decreasing apoptosis in patients with acute kidney injury.

Rescue of secretion of rare-disease-associated misfolded mutant glycoproteins in UGGT1 knock-out mammalian cells.

Endoplasmic reticulum (ER) retention of misfolded glycoproteins is mediated by the ER-localized eukaryotic glycoprotein secretion checkpoint, UDP-glucose glycoprotein glucosyl-transferase (UGGT). The enzyme recognizes a misfolded glycoprotein and flags it for ER retention by re-glucosylating one of its N-linked glycans. In the background of a congenital mutation in a secreted glycoprotein gene, UGGT-mediated ER retention can cause rare disease, even if the mutant glycoprotein retains activity ("responsive mutant"). Using confocal laser scanning microscopy, we investigated here the subcellular localization of the human Trop-2-Q118E, E227K and L186P mutants, which cause gelatinous drop-like corneal dystrophy (GDLD). Compared with the wild-type Trop-2, which is correctly localized at the plasma membrane, these Trop-2 mutants are retained in the ER. We studied fluorescent chimeras of the Trop-2 Q118E, E227K and L186P mutants in mammalian cells harboring CRISPR/Cas9-mediated inhibition of the UGGT1 and/or UGGT2 genes. The membrane localization of the Trop-2 Q118E, E227K and L186P mutants was successfully rescued in UGGT1-/- cells. UGGT1 also efficiently reglucosylated Trop-2-Q118E-EYFP in cellula. The study supports the hypothesis that UGGT1 modulation would constitute a novel therapeutic strategy for the treatment of pathological conditions associated to misfolded membrane glycoproteins (whenever the mutation impairs but does not abrogate function), and it encourages the testing of modulators of ER glycoprotein folding quality control as broad-spectrum rescue-of-secretion drugs in rare diseases caused by responsive secreted glycoprotein mutants.

Large scale plasma proteomics identifies novel proteins and protein networks associated with heart failure development.

Heart failure (HF) causes substantial morbidity and mortality but its pathobiology is incompletely understood. The proteome is a promising intermediate phenotype for discovery of novel mechanisms. We measured 4877 plasma proteins in 13,900 HF-free individuals across three analysis sets with diverse age, geography, and HF ascertainment to identify circulating proteins and protein networks associated with HF development. Parallel analyses in Atherosclerosis Risk in Communities study participants in mid-life and late-life and in Trøndelag Health Study participants identified 37 proteins consistently associated with incident HF independent of traditional risk factors. Mendelian randomization supported causal effects of 10 on HF, HF risk factors, or left ventricular size and function, including matricellular (e.g. SPON1, MFAP4), senescence-associated (FSTL3, IGFBP7), and inflammatory (SVEP1, CCL15, ITIH3) proteins. Protein co-regulation network analyses identified 5 modules associated with HF risk, two of which were influenced by genetic variants that implicated trans hotspots within the VTN and CFH genes.

Branchial CO2 and ammonia excretion in crustaceans: Involvement of an apical Rhesus-like glycoprotein.

AIM: To determine whether the crustacean Rh1 protein functions as a dual CO2 /ammonia transporter and investigate its role in branchial ammonia excretion and acid-base regulation. METHODS: Sequence analysis of decapod Rh1 proteins was used to determine the conservation of amino acid residues putatively involved in ammonia transport and CO2 binding in human and bacterial Rh proteins. Using the Carcinus maenas Rh1 protein (CmRh1) as a representative of decapod Rh1 proteins, we test the ammonia and CO2 transport capabilities of CmRh1 through heterologous expression in yeast and Xenopus oocytes coupled with site-directed mutagenesis. Quantitative PCR was used to assess the distribution of CmRh1 mRNA in various tissues. Western blotting was used to assess CmRh1 protein expression changes in response to high environmental ammonia and CO2 . Further, immunohistochemistry was used to assess sub-cellular localization of CmRh1 and a membrane-bound carbonic anhydrase (CmCAg). RESULTS: Sequence analysis of decapod Rh proteins revealed high conservation of several amino acid residues putatively involved in conducting ammonia transport and CO2 binding. Expression of CmRh1 in Xenopus oocytes enhanced both ammonia and CO2 transport which was nullified in CmRh1 D180N mutant oocytes. Transport of the ammonia analog methylamine by CmRh1 is dependent on both ionized and un-ionized ammonia/methylamine species. CmRh1 was co-localized with CmCAg to the apical membrane of the crustacean gill and only experienced decreased protein expression in the anterior gills when exposed to high environmental ammonia. CONCLUSION: CmRh1 is the first identified apical transporter-mediated route for ammonia and CO2 excretion in the crustacean gill. Our findings shed further light on the potential universality of dual ammonia and CO2 transport capacity of Rhesus glycoproteins in both vertebrates and invertebrates.

CCDC65, encoding a component of the axonemal Nexin-Dynein regulatory complex, is required for sperm flagellum structure in humans.

Sperm flagella share an evolutionary conserved microtubule-based structure with motile cilia expressed at the surface of several cell types, such as the airways epithelial cells. As a result, male infertility can be observed as an isolated condition or a syndromic trait, illustrated by Primary Cilia Dyskinesia (PCD). We report two unrelated patients showing multiple morphological abnormalities of the sperm flagella (MMAF) and carrying distinct homozygous truncating variants in the PCD-associated gene CCDC65. We characterized one of the identified variants (c.1208del; p.Asn403Ilefs*9), which induces the near absence of CCDC65 protein in patient sperm. In Chlamydomonas, CCDC65 ortholog (DRC2, FAP250) is a component of the Nexin-Dynein Regulatory complex (N-DRC), which interconnects microtubule doublets and coordinates dynein arms activity. In sperm cells from the patient, we also show the loss of GAS8, another component of the N-DRC, supporting a structural/functional link between the two proteins. Our work indicates that, similarly to ciliary axoneme, CCDC65 is required for sperm flagellum structure. Importantly, our work provides first evidence that mutations in the PCD-associated gene CCDC65 also cause asthenozoospermia.

Biallelic variants in the synaptic vesicle glycoprotein 2 A are associated with epileptic encephalopathy.

Synaptic Vesicle Glycoprotein 2 A (SV2A) is a membrane protein of synaptic vesicles and the binding site of antiepileptic drug levetiracetam. Biallelic Arg383Gln is reported in a family with intractable epilepsy earlier. Here, we report on the second family with early onset drug resistant epilepsy. We identified homozygous Arg289Ter variant by exome sequencing that segregated with the phenotype in the family. The affected children in these two families are normal at birth and developed recurrent seizures beginning in the second month of life and developed secondary failure of growth and development. Knock out mice models earlier had replicated the human phenotype observed in these two families. These findings support that biallelic loss of function variants in SV2A result in early onset intractable epilepsy in humans.

A genome-edited N. benthamiana line for industrial-scale production of recombinant glycoproteins with targeted N-glycosylation.

Control over glycosylation is an important quality parameter in recombinant protein production. Here, we demonstrate the generation of a marker-free genome edited Nicotiana benthamiana N-glycosylation mutant (NbXF-KO) carrying inactivated ß1,2-xylosyltransferase and α1,3-fucosyltransferase genes. The knockout of seven genes and their stable inheritance was confirmed by DNA sequencing. Mass spectrometric analyses showed the synthesis of N-glycans devoid of plant-specific ß1,2-xylose and core α 1,3-fucose on endogenous proteins and a series of recombinantly expressed glycoproteins with different complexities. Further transient glycan engineering towards more diverse human-type N-glycans resulted in the production of recombinant proteins decorated with ß1,4-galactosylated and α2,6-sialylated structures, respectively. Notably, a monoclonal antibody expressed in the NbXF-KO displayed glycosylation-dependent activities. Collectively, the engineered plants grow normally and are well suited for upscaling, thereby meeting industrial and regulatory requirements for the production of high-quality therapeutic proteins.

The down-regulation of STC2 mRNA may serve as a biomarker for death from mechanical asphyxia.

Death from mechanical asphyxia (DMA) is a common cause of death in forensic pathology. However, due to the lack of biomarkers, the authentication of DMA now relies on a series of non-specific signs, which may cause troubles in the judicial trials, especially when the criminal scene is not fully elucidated. To search for the potential biomarkers for DMA, brain samples of DMA and craniocerebral injury groups were screened by microarray. The obtained mRNAs were validated by animal and human samples. Primary cell culture was conducted to explore the biochemical changes under hypoxia. 415 differentially expressed mRNAs between two groups were discovered. Ten mRNAs were examined in both human and animal samples died of different causes of death. Stanniocalcin-2 (STC2) showed significant down-regulation in DMA samples compared to other groups, regardless of PMI, age, or temperature. Cellular experiments indicated that ROS level peaked after 15-min-hypoxic culture, when the expression level of STC2 was significant down-regulated simultaneously. The ER-stress-related proteins also showed potential connection with STC2. In general, it is indicated that the down-regulation of STC2 may serve as a biomarker for DMA.