Enkephalin-mediated modulation of basal somatic sensitivity by regulatory T cells in mice.

CD4+CD25+Foxp3+ regulatory T cells (Treg) have been implicated in pain modulation in various inflammatory conditions. However, whether Treg cells hamper pain at steady state and by which mechanism is still unclear. From a meta-analysis of the transcriptomes of murine Treg and conventional T cells (Tconv), we observe that the proenkephalin gene (Penk), encoding the precursor of analgesic opioid peptides, ranks among the top 25 genes most enriched in Treg cells. We then present various evidence suggesting that Penk is regulated in part by members of the Tumor Necrosis Factor Receptor (TNFR) family and the transcription factor Basic leucine zipper transcription faatf-like (BATF). Using mice in which the promoter activity of Penk can be tracked with a fluorescent reporter, we also show that Penk expression is mostly detected in Treg and activated Tconv in non-inflammatory conditions in the colon and skin. Functionally, Treg cells proficient or deficient for Penk suppress equally well the proliferation of effector T cells in vitro and autoimmune colitis in vivo. In contrast, inducible ablation of Penk in Treg leads to heat hyperalgesia in both male and female mice. Overall, our results indicate that Treg might play a key role at modulating basal somatic sensitivity in mice through the production of analgesic opioid peptides.

Both middle and large envelope proteins can mediate neutralization of hepatitis B virus infectivity by anti-preS2 antibodies: escape by naturally occurring preS2 deletions.

Hepatitis B virus (HBV) expresses co-terminal large (L), middle (M), and small (S) envelope proteins containing preS1/preS2/S, preS2/S, and S domain alone, respectively. S and preS1 domains mediate sequential virion attachment to heparan sulfate proteoglycans and sodium taurocholate cotransporting polypeptide (NTCP), respectively, which can be blocked by anti-S and anti-preS1 antibodies. How anti-preS2 antibodies neutralize HBV infectivity remains enigmatic. The late stage of chronic HBV infection often selects for mutated preS2 translation initiation codon to prevent M protein expression, or in-frame preS2 deletions to shorten both L and M proteins. When introduced to infectious clone of genotype C or D, both M-minus mutations and most 5' preS2 deletions sustained virion production. Such mutant progeny viral particles were infectious in NTCP-reconstituted HepG2 cells. Neutralization experiments were performed on the genotype D clone. Although remaining susceptible to anti-preS1 and anti-S neutralizing antibodies, M-minus mutants were only partially neutralized by two anti-preS2 antibodies tested while preS2 deletion mutants were resistant. By infection experiments using viral particles with lost versus increased M protein expression, or a neutralization escaping preS2 deletion only present on L or M protein, we found that both full-length L and M proteins contributed to virus neutralization by the two anti-preS2 antibodies. Thus, immune escape could be a driving force for the selection of M-minus mutations, and especially preS2 deletions. The fact that both L and M proteins could mediate neutralization by anti-preS2 antibodies may shed light on the underlying molecular mechanism.IMPORTANCEThe large (L), middle (M), and small (S) envelope proteins of hepatitis B virus (HBV) contain preS1/preS2/S, preS2/S, and S domain alone, respectively. The discovery of heparan sulfate proteoglycans and sodium taurocholate cotransporting polypeptide (NTCP) as the low- and high-affinity HBV receptors could explain neutralizing potential of anti-S and anti-preS1 antibodies, respectively, but how anti-preS2 neutralizing antibodies work remains enigmatic. In this study, we found two M-minus mutants in the context of genotype D partially escaped two anti-preS2 neutralizing antibodies in NTCP-reconstituted HepG2 cells, while several naturally occurring preS2 deletion mutants escaped both antibodies. By point mutations to eliminate or enhance M protein expression, and by introducing preS2 deletion selectively to L or M protein, we found binding of anti-preS2 antibodies to both L and M proteins contributed to neutralization of wild-type HBV infectivity. Our finding may shed light on the possible mechanism(s) whereby anti-preS2 antibodies neutralize HBV infectivity.

Evaluation of Kynu, Defb2, Camp, and Penk Expression Levels as Psoriasis Marker in the Imiquimod-Induced Psoriasis Model.

Background: Psoriasis is a noncontagious auto-inflammatory chronic skin disease. So far, some of the inflammatory genes were upregulated in mouse model of psoriasis. This study examined changes in skin mRNA expression of L-kynureninase (Kynu), cathelicidin antimicrobial peptide (Camp), beta-defensin 2 (Defb2), and proenkephalin (Penk) in a mouse model of imiquimod-induced psoriasis. Materials and Methods: Tree groups of C57BL/6 female mice were allocated. The imiquimod (IMQ) cream was administered to the mice dorsal skin of the two groups to induce psoriatic inflammation. In the treatment group, IMQ was administered 10 min after hydrogel-containing M7 anti-IL-17A aptamer treatment. Vaseline (Vas) was administered to the negative control group. The psoriatic skin lesions were evaluated based on the psoriasis area severity index (PASI) score, histopathology, and mRNA expression levels of Kynu, Camp, Defb2, and Penk using real-time PCR. In order to assess the systemic response, the spleen and lymph node indexes were also evaluated. Results: The PASI and epidermal thickness scores were 6.01 and 1.96, respectively, in the IMQ group, and they significantly decreased after aptamer administration to 1.15 and 0.90, respectively (P < 0.05). Spleen and lymph node indexes showed an increase in the IMQ group, followed by a slight decrease after aptamer treatment (P > 0.05). Additionally, the mRNA expression levels of Kynu, Defb2, Camp, and Penk genes in the IMQ-treated region showed a significant 2.70, 4.56, 3.29, and 2.61-fold increase relative to the Vas mice, respectively (P < 0.05). The aptamer-treated region exhibited a significant decrease in these gene expression levels (P < 0.05). A positive correlation was found between Kynu, Penk, and Camp expression levels and erythema, as well as Camp expression with PASI, scaling, and thickness (P < 0.05). Conclusion: According to our results, it seems that Kynu, Camp, and Penk can be considered appropriate markers for the evaluation of psoriasis in IMQ-induced psoriasis. Also, the anti-IL-17 aptamer downregulated these important genes in this mouse model.

Progerin forms an abnormal meshwork and has a dominant-negative effect on the nuclear lamina.

Progerin, the protein that causes Hutchinson-Gilford progeria syndrome, triggers nuclear membrane (NM) ruptures and blebs, but the mechanisms are unclear. We suspected that the expression of progerin changes the overall structure of the nuclear lamina. High-resolution microscopy of smooth muscle cells (SMCs) revealed that lamin A and lamin B1 form independent meshworks with uniformly spaced openings (~0.085 µm2). The expression of progerin in SMCs resulted in the formation of an irregular meshwork with clusters of large openings (up to 1.4 µm2). The expression of progerin acted in a dominant-negative fashion to disrupt the morphology of the endogenous lamin B1 meshwork, triggering irregularities and large openings that closely resembled the irregularities and openings in the progerin meshwork. These abnormal meshworks were strongly associated with NM ruptures and blebs. Of note, the progerin meshwork was markedly abnormal in nuclear blebs that were deficient in lamin B1 (~50% of all blebs). That observation suggested that higher levels of lamin B1 expression might normalize the progerin meshwork and prevent NM ruptures and blebs. Indeed, increased lamin B1 expression reversed the morphological abnormalities in the progerin meshwork and markedly reduced the frequency of NM ruptures and blebs. Thus, progerin expression disrupts the overall structure of the nuclear lamina, but that effect-along with NM ruptures and blebs-can be abrogated by increased lamin B1 expression.

Sphingolipid-Enriched Porcine Placental Extract Promotes the Expression of Structural Genes and Desquamation Enzyme Genes in Cultured Human Keratinocytes.

Porcine placental extract (PPE) is commonly used in various health foods and cosmetics. PPE use in cosmetics predominantly consist of the water-soluble fraction derived from the entire placenta. In this report, we examined the effect of the hydrophobic constituents of the PPE, specifically the sphingolipid-enriched fraction designated as the sphingolipid-enriched porcine placental extract (SLPPE), on the expression of genes associated with skin function in cultured normal human epidermal keratinocytes. Using quantitative RT-PCR (qRT-PCR) analysis, we found that SLPPE concentrations ranging from 25 to 100 µg/mL upregulated the gene expression of key components associated with the cornified envelope structure (filaggrin (FLG), involucrin (IVL) and loricrin (LOR)), cornification enzymes (transglutaminase 1 (TGM1) and TGM5) and the desquamation enzymes (kallikrein 5 (KLK5) and KLK7). Additionally, KLK5p and FLG protein (FLGp) were detected in the culture supernatants of keratinocytes treated with SLPPE at these concentrations. These findings suggest that SLPPE is possible to promote the cornification and desquamation in epidermal keratinocytes, and it may offer potential benefits in cosmetics.

Social Isolation Induces Changes in the Monoaminergic Signalling in the Rat Medial Prefrontal Cortex.

(1) Background: The effects of short-term social isolation during adulthood have not yet been fully established in rats behaviourally, and not at all transcriptomically in the medial prefrontal cortex (mPFC). (2) Methods: We measured the behavioural effects of housing adult male rats in pairs or alone for 10 days. We also used RNA sequencing to measure the accompanying gene expression alterations in the mPFC of male rats. (3) Results: The isolated animals exhibited reduced sociability and social novelty preference, but increased social interaction. There was no change in their aggression, anxiety, or depression-like activity. Transcriptomic analysis revealed a differential expression of 46 genes between the groups. The KEGG pathway analysis showed that differentially expressed genes are involved in neuroactive ligand-receptor interactions, particularly in the dopaminergic and peptidergic systems, and addiction. Subsequent validation confirmed the decreased level of three altered genes: regulator of G protein signalling 9 (Rgs9), serotonin receptor 2c (Htr2c), and Prodynorphin (Pdyn), which are involved in dopaminergic, serotonergic, and peptidergic function, respectively. Antagonizing Htr2c confirmed its role in social novelty discrimination. (4) Conclusions: Social homeostatic regulations include monoaminergic and peptidergic systems of the mPFC.

Estrogen promotes gonadotropin-releasing hormone expression by regulating tachykinin 3 and prodynorphin systems in chicken.

The "KNDy neurons" located in the hypothalamic arcuate nucleus (ARC) of mammals are known to co-express kisspeptin, neurokinin B (NKB), and dynorphin (DYN), and have been identified as key mediators of the feedback regulation of steroid hormones on gonadotropin-releasing hormone (GnRH). However, in birds, the genes encoding kisspeptin and its receptor GPR54 are genomic lost, leaving unclear mechanisms for feedback regulation of GnRH by steroid hormones. Here, the genes tachykinin 3 (TAC3) and prodynorphin (PDYN) encoding chicken NKB and DYN neuropeptides were successfully cloned. Temporal expression profiling indicated that TAC3, PDYN and their receptor genes (TACR3, OPRK1) were mainly expressed in the hypothalamus, with significantly higher expression at 30W than at 15W. Furthermore, overexpression or interference of TAC3 and PDYN can regulate the GnRH mRNA expression. In addition, in vivo and in vitro assays showed that estrogen (E2) could promote the mRNA expression of TAC3, PDYN, and GnRH, as well as the secretion of GnRH/LH. Mechanistically, E2 could dimerize the nuclear estrogen receptor 1 (ESR1) to regulate the expression of TAC3 and PDYN, which promoted the mRNA and protein expression of GnRH gene as well as the secretion of GnRH. In conclusion, these results revealed that E2 could regulate the GnRH expression through TAC3 and PDYN systems, providing novel insights for reproductive regulation in chickens.

A single-vector intersectional AAV strategy for interrogating cellular diversity and brain function.

As discovery of cellular diversity in the brain accelerates, so does the need for tools that target cells based on multiple features. Here we developed Conditional Viral Expression by Ribozyme Guided Degradation (ConVERGD), an adeno-associated virus-based, single-construct, intersectional targeting strategy that combines a self-cleaving ribozyme with traditional FLEx switches to deliver molecular cargo to specific neuronal subtypes. ConVERGD offers benefits over existing intersectional expression platforms, such as expanded intersectional targeting with up to five recombinase-based features, accommodation of larger and more complex payloads and a vector that is easy to modify for rapid toolkit expansion. In the present report we employed ConVERGD to characterize an unexplored subpopulation of norepinephrine (NE)-producing neurons within the rodent locus coeruleus that co-express the endogenous opioid gene prodynorphin (Pdyn). These studies showcase ConVERGD as a versatile tool for targeting diverse cell types and reveal Pdyn-expressing NE+ locus coeruleus neurons as a small neuronal subpopulation capable of driving anxiogenic behavioral responses in rodents.

Pre-proenkephalin 1 is Downregulated Under Unloading and is Involved in Osteoblast Biology.

Pre-proenkephalin 1 (Penk1) is a pro-neuropeptide that belongs to the typical opioid peptide's family, having analgesic properties. We previously found Penk1 to be the most downregulated gene in a whole gene profiling analysis performed in osteoblasts subjected to microgravity as a model of mechanical unloading. In this work, Penk1 downregulation was confirmed in the bones of two in vivo models of mechanical unloading: tail-suspended and botulinum toxin A (botox)-injected mice. Consistently, in the sera from healthy volunteers subjected to bed rest, we observed an inverse correlation between PENK1 and bed rest duration. These results prompted us to investigate a role for this factor in bone. Penk1 was highly expressed in mouse bone, but its global deletion failed to impact bone metabolism in vivo. Indeed, Penk1 knock out (Penk1-/-) mice did not show an overt bone phenotype compared to the WT littermates. Conversely, in vitro Penk1 gene expression progressively increased during osteoblast differentiation and its transient silencing in mature osteoblasts by siRNAs upregulated the transcription of the Sost1 gene encoding sclerostin, and decreased Wnt3a and Col1a1 mRNAs, suggesting an altered osteoblast activity due to an impairment of the Wnt pathway. In line with this, osteoblasts treated with the Penk1 encoded peptide, Met-enkephalin, showed an increase of Osx and Col1a1 mRNAs and enhanced nodule mineralization. Interestingly, primary osteoblasts isolated from Penk1-/- mice showed lower metabolic activity, ALP activity, and nodule mineralization, as well as a lower number of CFU-F compared to osteoblasts isolated from WT mice, suggesting that, unlike the transient inhibition, the chronic Penk1 deletion affects both osteoblast differentiation and activity. Taken together, these results highlight a role for Penk1 in the regulation of the response of the bone to mechanical unloading, potentially acting on osteoblast differentiation and activity in a cell-autonomous manner.

Effect of prothymosin α on neuroplasticity following cerebral ischemia­reperfusion injury.

Prothymosin α (ProT), a highly acidic nuclear protein with multiple cellular functions, has shown potential neuroprotective properties attributed to its anti­necrotic and anti­apoptotic activities. The present study aimed to investigate the beneficial effect of ProT on neuroplasticity after ischemia­reperfusion injury and elucidate its underlying mechanism of action. Primary cortical neurons were either treated with ProT or overexpressing ProT by gene transfection and exposed to oxygen­glucose deprivation for 2 h in vitro. Immunofluorescence staining for ProT and MAP­2 was performed to quantify ProT protein expression and assess neuronal arborization. Mice treated with vehicle or ProT (100 µg/kg) and ProT overexpression in transgenic mice received middle cerebral artery occlusion for 50 min to evaluate the effect of ProT on neuroplasticity­associated protein following ischemia­reperfusion injury. The results demonstrated that in cultured neurons ProT significantly increased neurite lengths and the number of branches, accompanied by an upregulation mRNA level of brain­derived neurotrophic factor. Furthermore, ProT administration improved the protein expressions of synaptosomal­associated protein, 25 kDa and postsynaptic density protein 95 after ischemic­reperfusion injury in vivo. These findings suggested that ProT can potentially induce neuroplasticity effects following ischemia­reperfusion injury.

Cysteine-free cone snail venom peptides: Classification of precursor proteins and identification of mature peptides.

The cysteine-free acyclic peptides present in marine cone snail venom have been much less investigated than their disulfide bonded counterparts. Precursor protein sequences derived from transcriptomic data, together with mass spectrometric fragmentation patterns for peptides present in venom duct tissue extracts, permit the identification of mature peptides. Twelve distinct gene superfamiles have been identified with precursor lengths between 64 and 158 residues. In the case of Conus monile, three distinct mature peptides have been identified, arising from two distinct protein precursors. Mature acyclic peptides are often post-translationally modified, with C-terminus amidation, a feature characteristic of neuropeptides. In the present study, 20 acyclic peptides from Conus monile and Conus betulinus were identified. The common modifications of C-terminus amidation, gamma carboxylation of glutamic acid (E to ϒ), N-terminus conversion of Gln (Q) to a pyroglutamyl residue (Z), and hydroxylation of Pro (P) to Hyp (O) are observed in one or more peptides identified in this study. Proteolytic trimming of sequences by cleavage at the C-terminus of Asn (N) residues is established. The presence of an asparagine endopeptidase is strengthened by the identification of legumain-like sequences in the transcriptome assemblies from diverse Conus species. Such sequences may be expected to have a cleavage specificity at Asn-Xxx peptide bonds.

Evaluating Prophylactic Effect of Bovine Colostrum on Intestinal Barrier Function in Zonulin Transgenic Mice: A Transcriptomic Study.

The intestinal barrier comprises a single layer of epithelial cells tightly joined to form a physical barrier. Disruption or compromise of the intestinal barrier can lead to the inadvertent activation of immune cells, potentially causing an increased risk of chronic inflammation in various tissues. Recent research has suggested that specific dietary components may influence the function of the intestinal barrier, potentially offering a means to prevent or mitigate inflammatory disorders. However, the precise mechanism underlying these effects remains unclear. Bovine colostrum (BC), the first milk from cows after calving, is a natural source of nutrients with immunomodulatory, anti-inflammatory, and gut-barrier fortifying properties. This novel study sought to investigate the transcriptome in BC-treated Zonulin transgenic mice (Ztm), characterized by dysbiotic microbiota, intestinal hyperpermeability, and mild hyperactivity, applying RNA sequencing. Seventy-five tissue samples from the duodenum, colon, and brain of Ztm and wild-type (WT) mice were dissected, processed, and RNA sequenced. The expression profiles were analyzed and integrated to identify differentially expressed genes (DEGs) and differentially expressed transcripts (DETs). These were then further examined using bioinformatics tools. RNA-seq analysis identified 1298 DEGs and 20,952 DETs in the paired (Ztm treatment vs. Ztm control) and reference (WT controls) groups. Of these, 733 DEGs and 10,476 DETs were upregulated, while 565 DEGs and 6097 DETs were downregulated. BC-treated Ztm female mice showed significant upregulation of cingulin (Cgn) and claudin 12 (Cldn12) duodenum and protein interactions, as well as molecular pathways and interactions pertaining to tight junctions, while BC-treated Ztm males displayed an upregulation of transcripts like occludin (Ocln) and Rho/Rac guanine nucleotide exchange factor 2 (Arhgf2) and cellular structures and interfaces, protein-protein interactions, and organization and response mechanisms. This comprehensive analysis reveals the influence of BC treatment on tight junctions (TJs) and Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) signaling pathway gene expressions. The present study is the first to analyze intestinal and brain samples from BC-treated Ztm mice applying high-throughput RNA sequencing. This study revealed molecular interaction in intestinal barrier function and identified hub genes and their functional pathways and biological processes in response to BC treatment in Ztm mice. Further research is needed to validate these findings and explore their implications for dietary interventions aimed at improving intestinal barrier integrity and function. The MGH Institutional Animal Care and Use Committee authorized the animal study (2013N000013).

Distinct neurexin-cerebellin complexes control AMPA- and NMDA-receptor responses in a circuit-dependent manner.

At CA1→subiculum synapses, alternatively spliced neurexin-1 (Nrxn1SS4+) and neurexin-3 (Nrxn3SS4+) enhance NMDA-receptors and suppress AMPA-receptors, respectively, without affecting synapse formation. Nrxn1SS4+ and Nrxn3SS4+ act by binding to secreted cerebellin-2 (Cbln2) that in turn activates postsynaptic GluD1 receptors. Whether neurexin-Cbln2-GluD1 signaling has additional functions besides regulating NMDA- and AMPA-receptors, and whether such signaling performs similar roles at other synapses, however, remains unknown. Here, we demonstrate using constitutive Cbln2 deletions in mice that at CA1→subiculum synapses, Cbln2 performs no additional developmental roles besides regulating AMPA- and NMDA-receptors. Moreover, low-level expression of functionally redundant Cbln1 did not compensate for a possible synapse-formation function of Cbln2 at CA1→subiculum synapses. In exploring the generality of these findings, we examined the prefrontal cortex where Cbln2 was recently implicated in spinogenesis, and the cerebellum where Cbln1 is known to regulate parallel-fiber synapses. In the prefrontal cortex, Nrxn1SS4+-Cbln2 signaling selectively controlled NMDA-receptors without affecting spine or synapse numbers, whereas Nrxn3SS4+-Cbln2 signaling had no apparent role. In the cerebellum, conversely, Nrxn3SS4+-Cbln1 signaling regulated AMPA-receptors, whereas now Nrxn1SS4+-Cbln1 signaling had no manifest effect. Thus, Nrxn1SS4+- and Nrxn3SS4+-Cbln1/2 signaling complexes differentially control NMDA- and AMPA-receptors in different synapses in diverse neural circuits without regulating synapse or spine formation.

Comprehensive evaluation of microRNA as a biomarker for the diagnosis of hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer. Current guidelines for HCC management recommend surveillance of high-risk patients every 6 mo using ultrasonography. Serum biomarkers, like alpha-fetoprotein (AFP), protein induced by vitamin K absence/antagonist-II (PIVKA-II) and lectin-reactive AFP, show suboptimal performance for detection of HCC, which is crucial for successful resection or treatment. Thus, there is a significant need for new biomarkers to aid early diagnosis of HCC. Studies have shown that the expression level of human microRNAs (miRNAs), a small, non-coding RNA species released into the blood, can serve as an early marker for various diseases, including HCC. AIM: To evaluate the diagnostic role of miRNAs in HCC as single markers, signatures or in combination with known protein biomarkers. METHODS: Our prospective, multicenter, case-control study recruited 660 participants (354 controls with chronic liver disease and 306 participants with HCC) and employed a strategy of initial screening by two independent methods, real-time quantitative PCR (n = 60) and next-generation sequencing (n = 100), to assess a large number of miRNAs. The results from the next-generation sequencing and real-time quantitative PCR screening approaches were then combined to select 26 miRNAs (including two putative novel miRNAs). Those miRNAs were analyzed for their diagnostic potential as single markers or in combination with other miRNAs or established protein biomarkers AFP and PIVKA-II via real-time quantitative PCR in training (n = 200) and validation cohorts (n = 300). RESULTS: We identified 26 miRNAs that differentiated chronic liver disease controls from (early) HCC via two independent discovery approaches. Three miRNAs, miR-21-5p (miR-21), miR-320a and miR-186-5p, were selected by both methods. In the training cohort, only miR-21, miR-320d and miR-423 could significantly distinguish (Q < 0.05) between the HCC and chronic liver disease control groups. In the multivariate setting, miR-21 with PIVKA-II was selected as the best combination, resulting in an area under the curve of 0.87 for diagnosis and area under the curve of 0.74 for early diagnosis of HCC. In the validation cohort, only miR-21 and miR-423 could be confirmed as potential HCC biomarkers. A combination of miRNAs did not perform better than any single miRNA. Improvement of PIVKA-II performance through combination with miRNAs could not be confirmed in the validation panel. Two putative miRs, put-miR-6 and put-miR-99, were tested in the training and validation panels, but their expression could only be detected in very few samples and at a low level (cycle threshold between 31.24 and 34.97). CONCLUSION: miRNAs alone or as a signature in combination with protein biomarkers AFP and PIVKA-II do not improve the diagnostic performance of the protein biomarkers.

Template-independent genome editing in the Pcdh15av-3j mouse, a model of human DFNB23 nonsyndromic deafness.

Although frameshift mutations lead to 22% of inherited Mendelian disorders in humans, there is no efficient in vivo gene therapy strategy available to date, particularly in nondividing cells. Here, we show that nonhomologous end-joining (NHEJ)-mediated nonrandom editing profiles compensate the frameshift mutation in the Pcdh15 gene and restore the lost mechanotransduction function in postmitotic hair cells of Pcdh15av-3J mice, an animal model of human nonsyndromic deafness DFNB23. Identified by an ex vivo evaluation system in cultured cochlear explants, the selected guide RNA restores reading frame in approximately 50% of indel products and recovers mechanotransduction in more than 70% of targeted hair cells. In vivo treatment shows that half of the animals gain improvements in auditory responses, and balance function is restored in the majority of injected mutant mice. These results demonstrate that NHEJ-mediated reading-frame restoration is a simple and efficient strategy in postmitotic systems.

Reporter system controlled by the involucrin promoter as a tool to follow epidermal differentiation.

Various approaches have been explored to study skin biology, including the use of stem cells. Mesenchymal stem cells (MSCs) from the umbilical cord can be safely and easily obtained; however, a simple strategy to monitor their differentiation is essential. Involucrin is a marker of keratinocyte differentiation, and its promoter (pINV) directs stratum-specific expression of this protein. We designed a reporter system containing EGFP under the control of pINV to assess MSC transdifferentiation into keratinocytes. The functional sequence of pINV was inserted into a lentiviral vector, producing LeGO-GpINV. MSCs were transduced with LeGO-GpINV and induced to transdifferentiate into keratinocytes under cultivation with keratinocyte serum-free medium. MSC transdifferentiation was confirmed by morphological changes and by the expression of epidermal markers by flow cytometry, quantitative PCR, Western blot and the activity of epidermal kallikreins 5, 6 and 7. After 14 days of transdifferentiation, MSCs transduced with LeGO-GpINV showed an increase in EGFP fluorescence and expressed CK10, CK14, involucrin and filaggrin. There was also an increase in kallikrein activity. This reporter system allowed us to temporally assess epidermal differentiation, simultaneously with involucrin expression, opening possibilities for the in vivo study of skin biology and in regenerative medicine.

Untangling the Conformational Plasticity of V66M Human proBDNF Polymorphism as a Modifier of Psychiatric Disorder Susceptibility.

The human genetic variant BDNF (V66M) represents the first example of neurotrophin family member that has been linked to psychiatric disorders. In order to elucidate structural differences that account for the effects in cognitive function, this hproBDNF polymorph was expressed, refolded, purified, and compared directly to the WT variant for the first time for differences in their 3D structures by DSF, limited proteolysis, FT-IR, and SAXS measurements in solution. Our complementary studies revealed a deep impact of V66M polymorphism on hproBDNF conformations in solution. Although the mean conformation in solution appears to be more compact in the V66M variant, overall, we demonstrated a large increase in flexibility in solution upon V66M mutation. Thus, considering that plasticity in IDR is crucial for protein function, the observed alterations may be related to the functional alterations in hproBDNF binding to its receptors p75NTR, sortilin, HAP1, and SorCS2. These effects can provoke altered intracellular neuronal trafficking and/or affect proBDNF physiological functions, leading to many brain-associated diseases and conditions such as cognitive impairment and anxiety. The structural alterations highlighted in the present study may pave the way to the development of drug discovery strategies to provide greater therapeutic responses and of novel pharmacologic strategy in human populations with this common polymorphism, ultimately guiding personalized medicine for neuropsychiatric disorders.

[PreS1 Antigen of HBV Down-Regulates MHC-â on the Surface of Hepatocytes and Promotes Hepatocarcinogenesis].

Objective: To explore the internal mechanism of hepatocellular carcinoma (HCC) induced by chronic hepatitis B virus (HBV) infection. Methods: L02, HepG2 and Huh7 cells stably overexpressing HBV preS1 antigen were analyzed by flow cytometry, qRT-PCR and tumorigenesis in nude mice to evaluate the effect of preS1 antigen in HBV-related hepatocarcinogenesis. Results: Our results showed that the expression of cancer stem cell (CSCs) related factors and cell surface markers in preS1 overexpressing cells were up-regulated, and the tumorigenicity of these cells was enhanced in nude mice. In addition, preS1 overexpression could down-regulate the expression of major histocompatibility complex Ⅰ (MHC-Ⅰ). The expression of MHC-Ⅰ on the cell surface could be restored by adding interferon gamma (IFN-γ) in the process of cell culturing and the tumorigenicity of cells in nude mice could thus be reduced. Conclusion: Based on the above results, we believe that preS1 is a carcinogen of HBV and that it promotes the formation of liver cancer through down regulating MHC-Ⅰ on the surface of hepatocytes.

Prothymosin Alpha: A Novel Contributor to Estradiol Receptor Alpha-Mediated CD8+ T-Cell Pathogenic Responses and Recognition of Type 1 Collagen in Rheumatic Heart Valve Disease.

BACKGROUND: Rheumatic heart valve disease (RHVD) is a leading cause of cardiovascular death in low- and middle-income countries and affects predominantly women. The underlying mechanisms of chronic valvular damage remain unexplored and regulators of sex predisposition are unknown. METHODS: Proteomics analysis of human heart valves (nondiseased aortic valves, nondiseased mitral valves [NDMVs], valves from patients with rheumatic aortic valve disease, and valves from patients with rheumatic mitral valve disease; n=30) followed by system biology analysis identified ProTα (prothymosin alpha) as a protein associated with RHVD. Histology, multiparameter flow cytometry, and enzyme-linked immunosorbent assay confirmed the expression of ProTα. In vitro experiments using peripheral mononuclear cells and valvular interstitial cells were performed using multiparameter flow cytometry and quantitative polymerase chain reaction. In silico analysis of the RHVD and Streptococcuspyogenes proteomes were used to identify mimic epitopes. RESULTS: A comparison of NDMV and nondiseased aortic valve proteomes established the baseline differences between nondiseased aortic and mitral valves. Thirteen unique proteins were enriched in NDMVs. Comparison of NDMVs versus valves from patients with rheumatic mitral valve disease and nondiseased aortic valves versus valves from patients with rheumatic aortic valve disease identified 213 proteins enriched in rheumatic valves. The expression of the 13 NDMV-enriched proteins was evaluated across the 213 proteins enriched in diseased valves, resulting in the discovery of ProTα common to valves from patients with rheumatic mitral valve disease and valves from patients with rheumatic aortic valve disease. ProTα plasma levels were significantly higher in patients with RHVD than in healthy individuals. Immunoreactive ProTα colocalized with CD8+ T cells in RHVD. Expression of ProTα and estrogen receptor alpha correlated strongly in circulating CD8+ T cells from patients with RHVD. Recombinant ProTα induced expression of the lytic proteins perforin and granzyme B by CD8+ T cells as well as higher estrogen receptor alpha expression. In addition, recombinant ProTα increased human leukocyte antigen class I levels in valvular interstitial cells. Treatment of CD8+ T cells with specific estrogen receptor alpha antagonist reduced the cytotoxic potential promoted by ProTα. In silico analysis of RHVD and Spyogenes proteomes revealed molecular mimicry between human type 1 collagen epitope and bacterial collagen-like protein, which induced CD8+ T-cell activation in vitro. CONCLUSIONS: ProTα-dependent CD8+ T-cell cytotoxicity was associated with estrogen receptor alpha activity, implicating ProTα as a potential regulator of sex predisposition in RHVD. ProTα facilitated recognition of type 1 collagen mimic epitopes by CD8+ T cells, suggesting mechanisms provoking autoimmunity.

A dominant negative variant of RAB5B disrupts maturation of surfactant protein B and surfactant protein C.

Pathogenic variants in surfactant proteins SP-B and SP-C cause surfactant deficiency and interstitial lung disease. Surfactant proteins are synthesized as precursors (proSP-B, proSP-C), trafficked, and processed via a vesicular-regulated secretion pathway; however, control of vesicular trafficking events is not fully understood. Through the Undiagnosed Diseases Network, we evaluated a child with interstitial lung disease suggestive of surfactant deficiency. Variants in known surfactant dysfunction disorder genes were not found in trio exome sequencing. Instead, a de novo heterozygous variant in RAB5B was identified in the Ras/Rab GTPases family nucleotide binding domain, p.Asp136His. Functional studies were performed in Caenorhabditis elegans by knocking the proband variant into the conserved position (Asp135) of the ortholog, rab-5 Genetic analysis demonstrated that rab-5[Asp135His] is damaging, producing a strong dominant negative gene product. rab-5[Asp135His] heterozygotes were also defective in endocytosis and early endosome (EE) fusion. Immunostaining studies of the proband's lung biopsy revealed that RAB5B and EE marker EEA1 were significantly reduced in alveolar type II cells and that mature SP-B and SP-C were significantly reduced, while proSP-B and proSP-C were normal. Furthermore, staining normal lung showed colocalization of RAB5B and EEA1 with proSP-B and proSP-C. These findings indicate that dominant negative-acting RAB5B Asp136His and EE dysfunction cause a defect in processing/trafficking to produce mature SP-B and SP-C, resulting in interstitial lung disease, and that RAB5B and EEs normally function in the surfactant secretion pathway. Together, the data suggest a noncanonical function for RAB5B and identify RAB5B p.Asp136His as a genetic mechanism for a surfactant dysfunction disorder.