The Prohibitin Protein Complex Promotes Mitochondrial Stabilization and Cell Survival In Hematologic Malignancies

Lymphocyte proliferation and differentiation is coordinated with high precision in healthy humans and is vital to maintaining a normal immune system. Imbalance of these events can result in the development of autoimmune diseases, immunodeficiencies and hematopoietic malignancies. These pathologies, specifically leukemia and lymphoma have a high incidence of relapse and mortality due to limited treatment options. Therefore, there is a critical need to characterize the signal transduction pathways and understand molecular hallmarks that mediate T cell activation in order to develop new strategies for diagnosis and treatments of these diseases. Prohibitins (PHB1 and PHB2) have been proposed to play important roles in cancer development and disease progression. In this study we used immunoprecipitation coupled to mass spectrometry to identify new forms of PHB regulation. We report four novel interleukin 2 (IL-2) inducible phosphosites in PHB2. To elucidate the potential regulatory role of these sites we generated phosphospecific polyclonal antibodies against one key phosphoresidue Threonine-62, to characterize its function. In addition, evidence is provided for PHB1 and PHB2 upregulation in tumor cell lines and localization mainly at the mitochondria. These proteins were also upregulated during reactive oxygen species (ROS)-mediated apoptosis. Similarity, PHB1 and PHB2 protein levels were significantly higher in tumor cells isolated from leukemia and lymphoma patients and determined to mainly localize to the mitochondria, possibly to maintain mitochondria integrity, which may facilitate the energy requirements of these tumor cells. Therefore, prohibitins may serve not only as biomarkers for cancer, but also act as molecular target for therapeutic intervention in hematopoietic malignancies

Rapid Shift from SARS-CoV-2 Delta to Omicron Sub-Variants within a Dynamic Southern U.S. Borderplex

COVID-19, caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), remains an ongoing global health challenge. This study analyzed 3641 SARS-CoV-2 positive samples from the El Paso, Texas, community and hospitalized patients over 48 weeks from Fall 2021 to Summer 2022. The binational community along the U.S. southern border was predominantly SARS-CoV-2 Delta variant (B.1.617.2) positive for a 5-week period from September 2021 to January 2022 and quickly transitioned to the Omicron variant (B.1.1.529), which was first detected at the end of December 2021. Omicron replaced Delta as the predominant detectable variant in the community and was associated with a sharp increase in COVID-19 positivity rate, related hospitalizations, and newly reported cases. In this study, Omicron BA.1, BA.4, and BA.5 variants were overwhelmingly associated with S-gene dropout by qRT-PCR analysis unlike the Delta and Omicron BA.2 variants. The study reveals that a dominant variant, like Delta, can be rapidly replaced by a more transmissible variant, like Omicron, within a dynamic metropolitan border city, necessitating enhanced monitoring, readiness, and response from public health officials and healthcare workers.</jats:p

The Genomic Landscape of a Restricted ALL Cohort from Patients Residing on the U.S./Mexico Border

Next-generation sequencing (NGS) has identified unique biomarkers yielding new strategies in precision medicine for the treatment of Acute lymphoblastic leukemia (ALL). Hispanics show marked health disparities in ALL, often absent in clinical trials or cancer research. Thus, it is unknown whether Hispanics would benefit equally from curated data currently guiding precision oncology. Using whole-exome sequencing, nine ALL patients were screened for mutations within genes known to possess diagnostic, prognostic and therapeutic value. Genes mutated in Hispanic ALL patients from the borderland were mined for potentially pathogenic variants within clinically relevant genes. KRAS G12A was detected in this unique cohort and its frequency in Hispanics from the TARGET-ALL Phase II database was three-fold greater than that of non-Hispanics. STAT5B N642H was also detected with low frequency in Hispanic and non-Hispanic individuals within TARGET. Its detection within this small cohort may reflect a common event in this demographic. Such variants occurring in the MAPK and JAK/STAT pathways may be contributing to Hispanic health disparities in ALL. Notable variants in ROS1, WT1, and NOTCH2 were observed in the ALL borderland cohort, with NOTCH2 C19W occurring most frequently. Further investigations on the pathogenicity of these variants are needed to assess their relevance in ALL

Rapid Shift from SARS-CoV-2 Delta to Omicron Sub-Variants within a Dynamic Southern U.S. Borderplex

COVID-19, caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), remains an ongoing global health challenge. This study analyzed 3641 SARS-CoV-2 positive samples from the El Paso, Texas, community and hospitalized patients over 48 weeks from Fall 2021 to Summer 2022. The binational community along the U.S. southern border was predominantly SARS-CoV-2 Delta variant (B.1.617.2) positive for a 5-week period from September 2021 to January 2022 and quickly transitioned to the Omicron variant (B.1.1.529), which was first detected at the end of December 2021. Omicron replaced Delta as the predominant detectable variant in the community and was associated with a sharp increase in COVID-19 positivity rate, related hospitalizations, and newly reported cases. In this study, Omicron BA.1, BA.4, and BA.5 variants were overwhelmingly associated with S-gene dropout by qRT-PCR analysis unlike the Delta and Omicron BA.2 variants. The study reveals that a dominant variant, like Delta, can be rapidly replaced by a more transmissible variant, like Omicron, within a dynamic metropolitan border city, necessitating enhanced monitoring, readiness, and response from public health officials and healthcare workers

The Genomic Landscape of a Restricted ALL Cohort from Patients Residing on the U.S./Mexico Border

Next-generation sequencing (NGS) has identified unique biomarkers yielding new strategies in precision medicine for the treatment of Acute lymphoblastic leukemia (ALL). Hispanics show marked health disparities in ALL, often absent in clinical trials or cancer research. Thus, it is unknown whether Hispanics would benefit equally from curated data currently guiding precision oncology. Using whole-exome sequencing, nine ALL patients were screened for mutations within genes known to possess diagnostic, prognostic and therapeutic value. Genes mutated in Hispanic ALL patients from the borderland were mined for potentially pathogenic variants within clinically relevant genes. KRAS G12A was detected in this unique cohort and its frequency in Hispanics from the TARGET-ALL Phase II database was three-fold greater than that of non-Hispanics. STAT5B N642H was also detected with low frequency in Hispanic and non-Hispanic individuals within TARGET. Its detection within this small cohort may reflect a common event in this demographic. Such variants occurring in the MAPK and JAK/STAT pathways may be contributing to Hispanic health disparities in ALL. Notable variants in ROS1, WT1, and NOTCH2 were observed in the ALL borderland cohort, with NOTCH2 C19W occurring most frequently. Further investigations on the pathogenicity of these variants are needed to assess their relevance in ALL.</jats:p

GB induced significant PS externalization on B-lymphoid lineage cells.

<p>The mode of inflicting cell death, apoptosis or necrosis, was monitored by flow cytometric assay after co-staining of the cells with annexin V-FITC and PI. Analysis was determined after 48 h and 96 h of incubation on (A–D) Nalm-6 and (A’–E’) BJAB cells, respectively. The total percentage of apoptotic cell populations is expressed as the sum of percentages of early and late stages of apoptosis (white bars), with green fluorescence signal indicating annexin V-FITC positive cells. Cells that were stained only with PI, due to the loss of plasma membrane integrity, were considered necrotic cells (black bars). Analysis of GB-treated Nalm-6 cells compared with PBS and untreated controls resulted in values of <i>P</i> < 0.00003 and <i>P</i> < 0.00002, respectively (**); whereas GB-treated BJAB cells as compared to PBS and untreated controls was <i>P</i> = 0.01284 and <i>P</i> = 0.00042 (*), respectively. Each bar represents the average of three replicas, and standard deviation. The following controls were included: cells exposed to 1 mg/ml G418, as a positive control for cytotoxicity; cells treated with 50 µl PBS; and untreated cells were also analyzed. Representative flow cytometric dot plots (A–D and B’–E’) that were used to determine the percentages of apoptosis/necrosis activity are depicted. Analysis of data from quadrant regions in the dot plots were interpreted as follows: the bottom left quadrant specifies unstained viable cells with intact membranes that are annexin V-FITC and PI double-negative; the top left quadrant denotes necrotic cells that are PI-positive and annexin V-FITC-negative; the top right quadrant includes late apoptotic cells that are annexin V-FITC and PI-positive; and the right bottom quadrant designates early apoptotic cells that are annexin V-FITC-positive, but PI-negative. The varied dot (event) color in each plot, designates just a density gradient; low-density region blue and high-density red. Approximately 1x10⁴ events were recorded and analyzed per individual sample using CXP software. GB 50 µl = 1.5 ± 0.048 mg/ml lyophilized powder.</p

Titanocene–Phosphine Derivatives as Precursors to Cytotoxic Heterometallic TiAu₂and TiM (M = Pd, Pt) Compounds. Studies of Their Interactions with DNA

A series of tri- and bimetallic titanium-gold, titanium-palladium and titanium-platinum derivatives of general formulas [Ti{η(5)-C(5)H(4)(CH(2))(n)PPh(2)(AuCl)}(2)].2THF n = 0 (1); n = 2 (2); n = 3 (3) and [TiCl(2){η(5)-C(5)H(4)κ-(CH(2))(n)PPh(2)}(2)(PtCl(2))].2THF (M = Pd, n = 0 (4); n = 2 (5); n = 3 (6); M = Pt, n = 0 (7); n = 2 (8); n = 3 (9)) have been synthesized and characterized by different spectroscopic techniques and mass spectrometry. The molecular structures of compounds 1–9 have been investigated by means of density-functional calculations. The calculated IR spectra of the optimized structures fit well with the experimental IR data obtained for 1–9. The stability of the heterometallic compounds in deuterated solvents (CDCl(3), d(6)-dmso, mixtures 50:50 d(6)-dmso/D(2)O, 1:99 d(6)-dmso/D(2)O at acidic pH and at neutral pH) has been evaluated by (31)P and (1)H NMR spectroscopy showing a higher stability for these compounds than for Cp(2)TiCl(2) or precursors [Ti{η(5)-C(5)H(4)(CH(2))(n)PPh(2)}(2)]. The new compounds display a lower acidity (1 to 2 units) than Cp(2)TiCl(2). The decomposition products have been identified over time. Complexes 1–9 have been tested as potential anticancer agents and their cytotoxicity properties were evaluated in vitro against HeLa human cervical carcinoma and DU-145 human prostate cancer cells. TiAu(2) and TiPd compounds were highly cytotoxic for these two cell lines. The interactions of the compounds with Calf Thymus DNA have been evaluated by Thermal Denaturation (1–9) and by Circular Dichroism (1, 3, 4, 7) spectroscopic methods. All these complexes show a stronger interaction with DNA than that displayed by Cp(2)TiCl(2) at neutral pH. The data is consistent with electrostatic interactions with DNA for TiAu(2) compounds and for a covalent binding mode for TiM (M = Pd, Pt) complexes

PKC-Dependent GlyT1 Ubiquitination Occurs Independent of Phosphorylation: Inespecificity in Lysine Selection for Ubiquitination

<div><p>Neurotransmitter transporter ubiquitination is emerging as the main mechanism for endocytosis and sorting of cargo into lysosomes. In this study, we demonstrate PKC-dependent ubiquitination of three different isoforms of the glycine transporter 1 (GlyT1). Incubation of cells expressing transporter with the PKC activator phorbol ester induced a dramatic, time-dependent increase in GlyT1 ubiquitination, followed by accumulation of GlyT1 in EEA1 positive early endosomes. This occurred via a mechanism that was abolished by inhibition of PKC. GlyT1 endocytosis was confirmed in both retinal sections and primary cultures of mouse amacrine neurons. Replacement of only all lysines in the <i>N</i>-and <i>C</i>-termini to arginines prevented ubiquitination and endocytosis, displaying redundancy in the mechanism of ubiquitination. Interestingly, a 40–50% reduction in glycine uptake was detected in phorbol-ester stimulated cells expressing the WT-GlyT1, whereas no significant change was for the mutant protein, demonstrating that endocytosis participates in the reduction of uptake. Consistent with previous findings for the dopamine transporter DAT, ubiquitination of GlyT1 tails functions as sorting signal to deliver transporter into the lysosome and removal of ubiquitination sites dramatically attenuated the rate of GlyT1 degradation. Finally, we showed for the first time that PKC-dependent GlyT1 phosphorylation was not affected by removal of ubiquitination sites, suggesting separate PKC-dependent signaling events for these posttranslational modifications.</p></div

Green barley mitigates cytotoxicity in human lymphocytes undergoing aggressive oxidative stress, via activation of both the Lyn/PI3K/Akt and MAPK/ERK pathways

Abstract Oxidative stress plays a critical role in numerous diseases. Therefore, the pursuit of compounds with antioxidant activity remains critical. Green barley young leaves aqueous extract (GB) was tested for its capacity to ameliorate cellular oxidative stress, and its potential cytoprotective mechanism was partially elucidated. Through Folin-Ciocalteau and 1,1-diphenyl-2-picrylhydrazyl (DPPH) colorimetric assays, GB total phenolic content and free radical scavenging activity were found to be 59.91 ± 2.17 mg/L and 110.75 µg/ml (IC50), respectively. Using a live cell-based propidium iodide dye exclusion assay and flow cytometry, GB was found to display significant cytoprotection activity on three human lymphocytic cell lines exposed to an aggressive H2O2-induced oxidative stress. The molecular mechanism for GB cytoprotection activity was assessed via bead-based xMAP technology on the Luminex platform and western blot analysis. GB treatment resulted in activation of Lyn, Akt, and ERK1/2, suggesting that GB is able to mitigate the H2O2-induced oxidative stress via activation of both the Lyn/PI3K/Akt and ERK/MAPK pathways. Our findings support the notion that GB extract has the potential to be a valuable therapeutic agent and may serve to establish a strategy to discover potential compound(s) or biological extracts/mixtures to be incorporated as a treatment to prevent oxidative stress-related diseases