Albumin Oxidation and Albumin Glycation Discordance During Type 2 Diabetes Therapy: Biological and Clinical Implications.

Aims: Cys34 albumin redox modifications (reversible "cysteinylation" and irreversible "di/trioxidation"), besides being just oxidative stress biomarkers, may have primary pathogenetic roles to initiate and/or aggravate cell, tissue, and vascular damage in diabetes. In an exploratory "proof-of-concept" pilot study, we examined longitudinal changes in albumin oxidation during diabetes therapy. Methods: Mass spectrometric analysis was utilized to monitor changes in human serum albumin (HSA) post-translational modifications {glycation [glycated albumin (GA)], cysteinylation [cysteinylated albumin (CA) or human non-mercaptalbumin-1; reversible], di/trioxidation (di/trioxidized albumin or human non-mercaptalbumin-2; irreversible), and truncation (truncated albumin)} during ongoing therapy. Four informative groups of subjects were evaluated [type 1 diabetes (T1DM), type 2 diabetes (T2DM), prediabetes-obesity, and healthy controls] at baseline, and subjects with diabetes were followed for a period up to 280 days. Results: At baseline, T2DM was associated with relatively enhanced albumin cysteinylation (CA% total) compared with T1DM (P = 0.004), despite comparable mean hyperglycemia (P values: hemoglobin A1c = 0.09; GA = 0.09). T2DM, compared with T1DM, exhibited selectively and significantly higher elevations of all the "individual" glycated cum cysteinylated ("multimodified") albumin isoforms (P values: CysHSA+1G = 0.003; CysHSA+2G = 0.007; and CysHSA+3G = 0.001). Improvements in glycemic control and decreases in albumin glycation during diabetes therapy in T2DM were not always associated with concurrent reductions of albumin cysteinylation, and in some therapeutic situations, albumin cysteinylation worsened (glycation-cysteinylation discordance). Important differences were observed between the effects of sulfonylureas and metformin on albumin molecular modifications. Conclusions: T2DM was associated with higher oxidative (cysteinylation) and combined (cysteinylation plus glycation) albumin molecular modifications, which are not ameliorated by improved glucose control alone. Further studies are required to establish the clinical significance and optimal therapeutic strategies to address oxidative protein damage and resulting consequences in diabetes.

Antiviral response and HIV-1 inhibition in sickle cell disease.

Sickle cell disease (SCD) is characterized by hemolysis, vaso-occlusion, and ischemia. HIV-1 infection was previously shown to be suppressed in SCD PBMCs. Here, we report that HIV-1 suppression is attributed to the increased expression of iron, hypoxia, and interferon-induced innate antiviral factors. Inhibition of upregulated antiviral genes, HMOX-1, CDKN1A, and CH25H, increased HIV-1 replication in SCD PBMCs, suggesting their critical role in HIV-1 suppression. Levels of IFN-ß were elevated in SCD patients. Sickle cell hemoglobin (HbS) treatment of THP-1-derived and primary monocyte-derived macrophages induced production of IFN-ß, upregulated antiviral gene expression, and suppressed HIV-1 infection. Infection with mouse-adapted EcoHIV was suppressed in the SCD mice that also exhibited elevated levels of antiviral restriction factors. Our findings suggest that hemolysis and release of HbS leads to the induction of IFN-ß production, induction of cellular antiviral state by the expression of iron and IFN-driven factors, and suppression of HIV-1 infection.

Assessment of risk of obstructive sleep apnea with thyroid eye disease and its activity.

PURPOSE: To evaluate the association between obstructive sleep apnea (OSA) and thyroid eye disease (TED) and its effect on disease activity. METHODS: A prospective case-control study was conducted from January 2020 to March 2022. All TED patients (group A) were clinically evaluated. The activity of thyroid eye disease was calculated based on the clinical activity score (CAS), and grading of severity was done according to the EUGOGO classification. All TED patients (group A) were screened for OSA using the Snoring Tired Observed Pressure (STOP)-Bang survey. Age- and gender-matched control group patients (group B) without TED were screened for OSA. RESULTS: One hundred TED patients and 138 control patients without TED were included in the respective groups. Sixty-two (62%) patients in group A and 48 (34.78%) patients in group B were having high risk of OSA, and this difference was statistically significant (P = 0.001). Further, in group A patients, on univariate analysis, TED activity was significantly associated with a high risk of OSA (P = 0.009). On multivariate logistic regression analysis, OSA also showed significant association with TED activity (odds ratio [OR]: 4.14, 95% confidence interval [CI]: 1.11-18.85 at 10% level; P = 0.05). CONCLUSION: Our study showed that OSA is significantly associated with TED disease and its activity. However, no significant association was found between OSA and severity of the disease.

Shear-reversible clusters of HIV-1 in solution: stabilized by antibodies, dispersed by mucin.

IMPORTANCE: The phenomenon of reversible clustering is expected to further nuance HIV immune stealth because virus surfaces can escape interaction with antibodies (Abs) by hiding temporarily within clusters. It is well known that mucin reduces HIV virulence, and the current perspective is that mucin aggregates HIV-1 to reduce infections. Our findings, however, suggest that mucin is dispersing HIV clusters. The study proposes a new paradigm for how HIV-1 may broadly evade Ab recognition with reversible clustering and why mucin effectively neutralizes HIV-1.

Differential Spectrum of Albumin Glycation, Oxidation, and Truncation in Type 2 and Type 1 Diabetes: Clinical and Biological Implications.

Background: Albumin, the most abundant and physiologically vital serum protein, accumulates a range of chemical modifications, as consequence of encounters with large number of reactive molecules whose concentrations increase in serum under pathological conditions. Methods: In a "proof of concept" study, mass spectrometric analysis was utilized to quantitate albumin post-translational modifications (glycation, oxidation, and truncation; individual isoforms and total) in four informative subject groups [type 1 diabetes (T1DM), type 2 diabetes (T2DM), prediabetes-obesity and healthy; all with estimated glomerular filtration rate ≥60 mL/(min·m2)]. Besides glycated albumin (GA/mass spectrometry), glycated serum protein (GSP/nitro blue tetrazolium colorimetry), and glycated hemoglobin (HbA1c/high-performance liquid chromatography) were also measured. Results: A wide spectrum of albumin molecular modifications was identified in diabetes, with significant differences between T2DM and T1DM. Albumin glycation: GA correlated more strongly with HbA1c in T1DM, compared to T2DM. Higher albumin glycation isoforms (human serum albumin +3G/2G) were more stable and discriminative markers of mean glycemia. Albumin oxidation: T2DM, in comparison with T1DM, showed enhanced oxidative and dual (glycation plus oxidation) modifications, representing extreme molecular pathology. Albumin truncation: There was dramatic reduction ("deficiency") of truncated albumin isoforms in T2DM, and significant reduction in T1DM. Albumin truncation negatively correlated with severity of albumin glycation (mean glycemia) and albumin oxidation (cysteinylation). Possible mechanisms of insulin resistance, with associated increased free fatty acids binding to albumin, in stimulating albumin oxidation and inhibiting albumin glycation ("metabolic cross talks") are reviewed. Conclusions: Albumin molecular modifications in diabetes, together with significant differences between T2DM and T1DM, suggest possible role for insulin resistance in their genesis and consequent cell, tissue, and vascular dysfunction/damage. Albumin molecular fingerprinting can provide valuable insights into pathogenesis, diagnosis, monitoring, and future therapies for diabetes. Identification of biomarker battery ("albuminomics," "diabetomics") driven diverse "healthy," prediabetes, obesity, and T2DM phenotypes represents additional novel step toward precision medicine in diabetes and related disorders.

Induction of Hepcidin Expression in the Renal Cortex of Sickle Cell Disease Mice.

In patients with sickle cell disease (SCD), chronic hemolysis and frequent blood transfusions cause iron overload and accumulation in the kidneys. The iron deposition is found in the renal cortex and correlates with the severity of hemolysis. In this study, we observed a significant accumulation of iron in the renal cortex of a mouse model of SCD, and assessed the expression of the proteins involved in maintaining renal iron homeostasis. Despite the intracellular iron accumulation, the levels of the transferrin receptor in the kidneys were increased, but the levels of the iron exporter ferroportin were not altered in SCD mice. Ferroportin is regulated by hepcidin, which binds to it and promotes its degradation. We found reduced serum hepcidin levels but increased renal hepcidin production in SCD mice. Furthermore, we observed significant macrophage infiltration and increased expression of intercellular adhesion molecule 1 in the endothelial cells of the kidneys in SCD mice. These observations correlated with elevated levels of proinflammatory cytokines IL-1ß and IL-6, which can potentially stimulate hepcidin expression. Taken together, our results demonstrate that in individuals with SCD, a renal inflammation state induces renal hepcidin production that blocks the upregulation of ferroportin levels, resulting in dysregulation of iron homeostasis in the kidney and iron deposition in the renal cortex.

Development and validation of most efficient RNA isolation method from buffalo bull spermatozoa.

BACKGROUND: Being highly fragmented and low in concentration, isolation of good quality RNA from sperm cells is a big challenge. Attempts have been made to evaluate various sperm RNA isolation methods from purified buffalo bull sperm cells. METHODS: Both, non-membrane and membrane-based methods have been evaluated for isolating RNA from Murrah buffalo sperms and compared for their respective efficacies. The traditional TRIzol, TRIzol-heat lysed (H-TRIzol) and cocktail of TCEP-RLT lysis buffer (Qiagen RNeasy mini kit)-TRIzol (C-TRIzol) based isopropanol isolation methods have been evaluated. RESULTS: H-TRIzol yielded best results among conventional methods. The combined T-RLT RNA isolation protocol yielded best quality and quantity compared to other membrane-based methods, due to high lytic property of cocktail of lysis reagents, necessary for complete breakdown of sperm membrane and RNA binding membrane for RNA isolation. Combined lysis performed by treatment with RLT-T and T-RLT differing in order of reagents used were also evaluated. T-RLT combination giving better results compared to RLT-T due to high gDNA contamination and membrane clogging in later protocol steps. CONCLUSION: Overall, in terms of total RNA quantity and quality per million spermatozoa, the heat-lysed TRIzol method (H-TRIzol) performs best among RNA separation techniques employed and is also quite easy to perform. This comparative evaluation of sperm RNA isolation protocols can be useful in deciding the best protocol for isolation of good quality and high concentration sperm RNA from buffalo semen, for transcriptome and other downstream studies.

Ebola Virus NP Binding to Host Protein Phosphatase-1 Regulates Capsid Formation.

The Ebola virus (EBOV) transcriptional regulation involves host protein phosphatases PP1 and PP2A, which dephosphorylate the transcriptional cofactor of EBOV polymerase VP30. The 1E7-03 compound, which targets PP1, induces VP30 phosphorylation and inhibits EBOV infection. This study aimed to investigate the role of PP1 in EBOV replication. When EBOV-infected cells were continuously treated with 1E7-03, the NP E619K mutation was selected. This mutation moderately reduced EBOV minigenome transcription, which was restored by the treatment with 1E7-03. Formation of EBOV capsids, when NP was co-expressed with VP24 and VP35, was impaired with NPE 619K. Treatment with 1E7-03 restored capsid formation by NP E619K mutation, but inhibited capsids formed by WT NP. The dimerization of NP E619K, tested in a split NanoBiT assay, was significantly decreased (~ 15-fold) compared to WT NP. NP E619K bound more efficiently to PP1 (~ 3-fold) but not B56 subunit of PP2A or VP30. Cross-linking and co-immunoprecipitation experiments showed fewer monomers and dimers for NP E619K which were increased with 1E7-03 treatment. NP E619K showed increased co-localization with PP1α compared to WT NP. Mutations of potential PP1 binding sites and NP deletions disrupted its interaction with PP1. Collectively, our findings suggest that PP1 binding to the NP regulates NP dimerization and capsid formation, and that NP E619K mutation, which has the enhanced PP1 binding, disrupts these processes. Our results point to a new role for PP1 in EBOV replication in which NP binding to PP1 may facilitate viral transcription by delaying capsid formation and EBOV replication.

ASIP gene polymorphism associated with black coat and skin color in Murrah buffalo.

The melanogenesis pathway regulates pigmentation through the synergic action of various genes. We are interested in analyzing the genetic variations in the ASIP which determine eumelanin production in the dermis layer. In the present study, the ASIP gene was characterized in buffalo and 268 genetically unrelated buffaloes belonging to 10 different populations were genotyped for the non-synonymous SNP (c.292C>T) identified in the exon 3 region of the gene using Tetra-ARMS-PCR. The TT genotype occurred at a higher rate in Murrah, followed by Nili Ravi, Tripura, and Paralakhemundi (42.63%, 19.30%, 3.45%, and 3.33%). These results convey the association of the black coat color of Murrah with the ASIP gene TT genotype and the lighter shades of black coat (brown and grayish-black) color phenotype in other breeds with the CC genotype.

Intra-arterial chemotherapy in refractory and advanced intraocular retinoblastoma.

Purpose: To evaluate the efficacy of secondary and salvage intra-arterial chemotherapy (IAC) as a globe salvage treatment modality in advanced and refractory intraocular retinoblastoma. Methods: A retrospective chart review of advanced intraocular retinoblastoma (groups D and E International Classification of Retinoblastoma [ICRB] classification) patients refractory to intravenous chemotherapy (IVC) and undergoing IAC as the secondary and salvage treatment modality between December 2018 and June 2021 was carried out. All patients underwent the IAC procedure by super-selective ophthalmic artery catheterization and with triple-drug chemotherapeutic agents of melphalan, topotecan, and carboplatin. Data were collected about tumor regression, eye salvage, metastasis, and survival outcome at follow-up. Results: Out of 13 patients, 12 patients received secondary IAC after being primarily treated with IVC and focal therapies and one patient received rescue IAC after recurrence following primary IAC. Mean number of IAC cycles administered was 2. Overall, globe salvage rate was 53.84%, with a mean follow-up of 17.53 months (range 6-37 months), three patients had enucleation for residual tumor or tumor recurrence. One patient developed metastasis post enucleation and two patients who were lost to follow-up after enucleation advice for residual tumor developed orbital tumor extension and eventually died of metastasis. Conclusion: Secondary triple-drug IAC following failure of IVC, along with other adjunct treatment modalities might a be a cost-effective option for eye salvage in advanced intraocular retinoblastoma patients who refuse enucleation, with a globe salvage rate of 53.84%. It can also be an effective approach to improve treatment compliance and can help in addressing the barrier of treatment refusal when enucleation is advised.

Allelic diversity at BoLA DRB3 locus and association with predisposition to clinical mastitis in indicus and crossbred cattle.

Bovine lymphocyte antigen (BoLA) DRB3 locus in healthy and mastitis affected cattle has been genotyped by a polymerase chain reaction and restriction fragment length polymorphisms (PCR-RLFP) using RsaI restriction enzyme, followed by sequencing. In 130 farm animals, 25 BoLA DRB3 alleles have been detected by PCR-RFLP. Three distinct allelic patterns significantly associated with mastitis in Karan Fries crossbred and Sahiwal indicus cattle have been identified, whereas, four other allelic patterns were significantly high in frequency among healthy animals. Sequencing of RFLP genotypes revealed 25 and 47 alleles among healthy Sahiwal and Karan Fries, respectively, while 17 and 38 patterns observed in mastitis affected Sahiwal and Karan Fries animals, respectively. From Tajima's D-test of neutrality, it was concluded that alleles associated with mastitis were expanding in the population, whereas those of healthy were under contraction. Phylogenetic analysis carried out to delineate the evolutionary relationship of the farm and field animals at DRB3 locus, differentiating allelic patterns into six different clusters. Among the phylogenetic lineages, five patterns DRB3*028:01, DRB3*011:03, DRB3*031:01, DRB3*001:01 and DRB3*043:01, were previously reported, whereas one novel allelic variant was observed in indicus and crossbred cattle. This information will help in further exploring the association between BoLA-DRB3 genetic diversity and disease resistance in distinct cattle breeds, important in designing breeding strategies for increasing the distribution of favorable alleles.

Primary orbital yolk sac tumor presenting as fungating mass.

Primary yolk sac tumor of the orbit is a rare entity. Orbital involvement is usually seen in young children and proptosis is the commonest presentation. Aggressive orbital involvement and presentation as a fungating mass is rarely seen. We report a case of primary orbital yolk sac tumor with an aggressive presentation that responded well to systemic chemotherapy.

HIV-1 Transcription Inhibitor 1E7-03 Decreases Nucleophosmin Phosphorylation.

Transcription activation of latent human immunodeficiency virus-1 (HIV-1) occurs due to HIV-1 rebound, the interruption of combination antiretroviral therapy, or development of drug resistance. Thus, novel HIV-1 inhibitors, targeting HIV-1 transcription are needed. We previously developed an HIV-1 transcription inhibitor, 1E7-03, that binds to the noncatalytic RVxF-accommodating site of protein phosphatase 1 and inhibits HIV-1 replication in cultured cells and HIV-1-infected humanized mice by impeding protein phosphatase 1 interaction with HIV-1 Tat protein. However, host proteins and regulatory pathways targeted by 1E7-03 that contribute to its overall HIV-1 inhibitory activity remain to be identified. To address this issue, we performed label-free quantitative proteome and phosphoproteome analyses of noninfected and HIV-1-infected CEM T cells that were untreated or treated with 1E7-03. 1E7-03 significantly reprogramed the phosphorylation profile of proteins including PPARα/RXRα, TGF-ß, and PKR pathways. Phosphorylation of nucleophosmin (NPM1) at Ser-125 residue in PPARα/RXRα pathway was significantly reduced (>20-fold, p = 1.37 × 10-9), followed by the reduced phosphorylation of transforming growth factor-beta 2 at Ser-46 (TGF-ß2, >12-fold, p = 1.37 × 10-3). Downregulation of NPM1's Ser-125 phosphorylation was further confirmed using Western blot. Phosphorylation mimicking NPM1 S125D mutant activated Tat-induced HIV-1 transcription and exhibited enhanced NPM1-Tat interaction compared to NPM1 S125A mutant. Inhibition of Aurora A or Aurora B kinases that phosphorylate NPM1 on Ser-125 residue inhibited HIV-1, further supporting the role of NPM1 in HIV-1 infection. Taken together, 1E7-03 reprogrammed PPARα/RXRα and TGF-ß pathways that contribute to the inhibition of HIV-1 transcription. Our findings suggest that NPM1 phosphorylation is a plausible target for HIV-1 transcription inhibition.

Monitoring Local pH of Membranous Aggregates via Ratiometric Color Changing Response.

A series of oxidized di(indolyl)arylmethanes (DIAM) with polyaromatic signaling moieties have been designed for monitoring local pH at the interfacial region of surfactant aggregates, such as micelles and vesicles. The oxidized DIAMs show changes in solution color from red to yellow when incorporated in cationic surfactants (at pH 7.4) and yellow to reddish pink when exposed to negatively-charged surfactants (at pH 5.0). The changes in surface charge can influence the interfacial pH (distinct from bulk pH of the medium) of the surfactant aggregates. The mechanistic studies indicate that the red-shifted absorption maxima observed in the presence of anionic amphiphiles (acidic local pH) originated from the protonated species. On the contrary, maxima in the blue region, triggered by positively charged amphiphiles (basic local pH), is attributed to the zwitterionic species. Such prototropic equilibrium affects charge transfer states of the molecules along with their self-assembly properties. Thus, it is evident that probes can predict as well as quantify the local pH change using the pseudophase ion exchange formalism. Also, the probes can detect the presence of anionic amphiphiles even when bound to phospholipid membranes.

HIV-1 infection in sickle cell disease and sickle cell trait: role of iron and innate response.

INTRODUCTION: Sickle cell disease (SCD), an inherited hemoglobinopathy, affects primarily African Americans in the U.S.A. In addition, about 15% African Americans carry sickle cell trait (SCT). Despite the risk associated with blood transfusions, SCD patients have lower risk of acquiring HIV-1 infection. SCT individuals might also have some protection from HIV-1 infection. AREAS COVERED: Here, we will review recent and previous studies with the focus on molecular mechanisms that might underlie and contribute to the protection of individuals with SCD and SCT from HIV-1 infection. As both of these conditions predispose to hemolysis, we will focus our discussion on the effects of systemic and intracellular iron on HIV-1 infection and progression. We will also review changes in iron metabolism and activation of innate antiviral responses in SCD and SCT and their effects on HIV-1 infection. EXPERT OPINION: Previous studies, including ours, showed that SCD might protect from HIV-1 infection. This protection is likely due to the upregulation of complex protein network in response to hemolysis, hypoxia and interferon signaling. These findings are important not only for HIV-1 field but also for SCD cure efforts as antiviral state of SCD patients may adversely affect lentivirus-based gene therapy efforts.

Identification of novel allelic patterns and evolutionary lineage of BoLA MHC class II DQA locus in indicine and taurine cattle.

Among different cattle types, Bos indicus are known for their ability to better resist the tropical microbial infections comparatively, wherein MHC molecules play a significant role. In this study allelic diversity at MHC locus, DQA of Bos indicus, Bos taurus and crossbred of taurine-indicus has been explored to understand the possible role of MHC region in differential immune response. Thirty nine different DQA alleles were identified, out of which 14 were novel, along with documentation of duplication of DQA alleles. Indicus cattle population presented diverse types of DQA alleles compared to crossbred and exotic. Translated amino acid sequence analysis indicated, codon 64 and 50 of peptide binding sites being highly polymorphic and most of the indicus cattle presented alanine and arginine amino acid at position 64 and 50. Within breed genetic variation found to be higher than between breeds. Because of their ability to bind and subsequently respond to a wide array of antigens, the newly identified DQA alleles with high diversity present in the form of duplicated haplotypes in different combinations in cattle populations provided significant insights into probable role of this MHC locus in better tropical disease combating ability and genetic fitness of indicus cattle.

RASAL2 suppresses the proliferative and invasive ability of PC3 prostate cancer cells.

The RAS protein activator like 2 (RASAL2) negatively regulates RAS proto-oncogene which is activated by high mutation rate in cancer. Thus, RASAL2 expression could potentially limit the function of RAS in prostate cancer (PCa). Genome-wide DNA methylation analysis demonstrated that RASAL2 is differentially hypermethylated in PCa tissues compared to benign prostate tissues. The PCR analysis of RASAL2 mRNA transcript showed differential expression in a panel of prostate cell lines with most PCa showing lower RASAL2 expression compared to benign prostatic epithelial cells. In PCa PC3 cells, the ectopic expression of RASAL2 significantly inhibited cell proliferation and invasion and induced an S phase plus G2/M phase cell cycle arrest. Ingenuity Pathway Analysis (IPA) demonstrated a cross talk between RASAL2 and TNFα, a key cytokine in immune signaling pathway that is relevant in PCa. Over-expression of RASAL2 downregulated TNFα expression whereas the knockdown of RASAL2 caused increased expression of TNFα. Taken together, our data demonstrates tumor suppressor role for RASAL2 in human PCa cells, despite increased RAS oncogenic activity. Our observation provides a new mechanistic insight of RASAL2 expression in aberrant Ras expression and immune signaling in PCa cells suggesting a potential novel therapeutic target for PCa.