Isolation and purification of total RNA from Streptococcus mutans in suspension cultures and biofilms

The presence of extracellular polysaccharides matrix makes extraction and purification of RNA from Streptococcus mutans within biofilms challenging. In this study, several approaches to purify RNA extracted from S. mutans in suspension cultures and biofilms were examined. The combination of sonication (3 pulses of 30 s at 7 W), suspension in NAES buffer (50 mM sodium acetate buffer, 10 mM EDTA and 1% SDS; pH 5.0) and homogenization-mechanical cells disruption in NAES- acid phenol:chloroform, yielded 9.04 mg (or 0.52 mg) of crude preparation of RNA per 100 mg of total cell (or biofilm) dry-weight. The crude RNA preparations were subjected to various DNAse I treatments. The combination of DNAse I in silica-gel based column followed by recombinant DNase I in solution provided the best genomic DNA removal, resulting in 4.35 mg (or 0.06 mg) of purified RNA per 100 mg of total cell (or biofilm) dry-weight. The cDNAs generated from the purified RNA sample were efficiently amplified using gtfB S. mutans-specific primers. The results showed a method that yields high-quality RNA from both planktonic cells and biofilms of S. mutans in sufficient quantity and quality for real-time RT-PCR analyses

Perceived Exertion during Moderate and Vigorous Physical Activity While Mask Wearing: A Quantitative and Qualitative Pilot Study

There is limited research examining the perception of exertion during exercise while wearing a facemask. The current study examined if mask usage during moderate or vigorous physical activity (MVPA) changed the self-reported perception of exertion. Seventy-two adults (18 years and older) who were physically active before the COVID-19 pandemic completed a questionnaire that assessed exercise habits and perceptions of mask wearing during MVPA. Participants reported their ratings of perceived exertion (RPE, on a scale of 1–10) while exercising. Wearing a mask resulted in higher RPE vs. no mask during both vigorous (8.4 ± 0.2 vs. 7.4 ± 0.1; p &lt; 0.001) and moderate PA (6.6 ± 0.2 vs. 5.6 ± 0.2; p &lt; 0.001). Qualitative analysis revealed mostly negative perceptions of exercising while wearing a mask, including respiratory issues, detriments to cardiovascular endurance, and general discomfort. A total of 40% of participants reported that they stopped exercising in an indoor/public setting because of a mask mandate in their region. Participants reported participating in less vigorous PA (4.7 ± 0.4 vs. 4.0 ± 0.4 h/week; p = 0.046), but not less moderate PA (3.3 ± 0.3 vs. 3.0 ± 0.3 h/week; p = 0.443) pre vs. during the pandemic. Our study suggests that facemask usage during MVPA causes an increase in RPE and may be one reason for a decrease in vigorous PA during the COVID-19 pandemic.</jats:p

Perceived Exertion during Moderate and Vigorous Physical Activity While Mask Wearing: A Quantitative and Qualitative Pilot Study

There is limited research examining the perception of exertion during exercise while wearing a facemask. The current study examined if mask usage during moderate or vigorous physical activity (MVPA) changed the self-reported perception of exertion. Seventy-two adults (18 years and older) who were physically active before the COVID-19 pandemic completed a questionnaire that assessed exercise habits and perceptions of mask wearing during MVPA. Participants reported their ratings of perceived exertion (RPE, on a scale of 1–10) while exercising. Wearing a mask resulted in higher RPE vs. no mask during both vigorous (8.4 ± 0.2 vs. 7.4 ± 0.1; p \u3c 0.001) and moderate PA (6.6 ± 0.2 vs. 5.6 ± 0.2; p \u3c 0.001). Qualitative analysis revealed mostly negative perceptions of exercising while wearing a mask, including respiratory issues, detriments to cardiovascular endurance, and general discomfort. A total of 40% of participants reported that they stopped exercising in an indoor/public setting because of a mask mandate in their region. Participants reported participating in less vigorous PA (4.7 ± 0.4 vs. 4.0 ± 0.4 h/week; p = 0.046), but not less moderate PA (3.3 ± 0.3 vs. 3.0 ± 0.3 h/week; p = 0.443) pre vs. during the pandemic. Our study suggests that facemask usage during MVPA causes an increase in RPE and may be one reason for a decrease in vigorous PA during the COVID-19 pandemic

Recent Advances in Understanding Cholangiocarcinoma [version 1; referees: 2 approved]

Cholangiocarcinoma (CCA) is an aggressive malignancy that arises from damaged epithelial cells, cholangiocytes, and possibly de-differentiated hepatocytes. CCA has a poor overall survival rate and limited therapeutic options. Based on this data, it is imperative that new diagnostic and therapeutic interventions be developed. Recent work has attempted to understand the pathological mechanisms driving CCA progression. Specifically, recent publications have delved into the role of cancer stem cells (CSCs), mesenchymal stem cells (MSCs), and microRNAs (miRNAs) during CCA pathology. CSCs are a specific subset of cells within the tumor environment that are derived from a cell with stem-like properties and have been shown to influence recurrence and chemoresistance during CCA. MSCs are known for their anti-inflammatory activity and have been postulated to influence malignancy during CCA, but little is known about their exact functions. miRNAs exert various functions via gene regulation at both the transcriptional and the translational levels, giving miRNAs diverse roles in CCA progression. Additionally, current miRNA-based therapeutic approaches are in clinical trials for various liver diseases, giving hope for similar approaches for CCA. However, the interactions among these three factors in the context of CCA are unknown. In this review, we focus on recently published data (within the last 3 years) that discuss the role of CSCs, MSCs, and miRNAs and their possible interactions during CCA pathogenesis

Influence of Apigenin on gtf Gene Expression in Streptococcus mutans UA159

Apigenin, a potent inhibitor of glucosyltransferase activity, affects the accumulation of Streptococcus mutans biofilms in vitro by reducing the formation of insoluble glucans and enhancing the soluble glucan content of the polysaccharide matrix. In the present study, we investigated the influence of apigenin on gtfB, gtfC, and gtfD expression in S. mutans UA159. Apigenin (0.1 mM) significantly decreased the expression of gtfB and gtfC mRNA (P < 0.05); in contrast, it increased the expression of gtfD in S. mutans growing in the planktonic state. The protein levels of GTF B, GTF C, and GTF D in culture supernatants were also affected; less GTF B and C were detected, whereas the level of GTF D was significantly elevated (P < 0.05). A similar profile of gtf expression was obtained with biofilms, although an elevated concentration (1 mM) of apigenin was required to elicit the effects. The influence of apigenin on gtf gene expression was independent of any effect on GTF activity, did not involve inhibition of growth or effects on pH, and was not affected by addition of sucrose. The data show that apigenin modulates the genetic expression of virulence factors in S. mutans

Abstract 936: Nonclinical safety and pharmacology of VX15/2503: a humanized IgG4 monoclonal antibody to SEMA4D

Abstract Vaccinex is developing VX15/2503, a humanized IgG4 anti-Semaphorin 4D (SEMA4D; CD100) antibody with approximately 5 nM affinity, for the treatment of patients with advanced solid tumors and for the treatment of patients with multiple sclerosis. SEMA4D is an important mediator of axonal growth cone guidance, angiogenesis, and T cell and B cell activation. Cellular SEMA4D (cSEMA4D) is expressed most abundantly on T cells and to a lesser extent on other lymphocytes as a 150 kDa transmembrane protein. SEMA4D is less abundantly present in serum as a 240 kDa, homodimeric soluble protein (sSEMA4D). VX15/2503 recognizes a discontinuous epitope in the homodimerzation domain of both the cellular and soluble forms of SEMA4D. This epitope is also located on the margin of the plexin B1 (PLXNB1) binding domain, the high affinity receptor of SEMA4D. Binding of SEMA4D by VX15/2503 blocks the binding of SEMA4D to PLXNB1 and reduces cSEMA4D by promoting internalization of approximately 60% over 24 hours. Evaluation of VX15/2503 or its murine progenitor (MAb 67-2) demonstrated that these anti-SEMA4D antibodies suppressed tumor growth, metastases and angiogenesis in syngeneic, xenograft, and transgenic tumor models. Single dose and one month toxicology studies utilizing VX15/2503 were performed in Sprague Dawley rats and cynomolgus macaques at doses of 0, 10, 30, or 100 mg/kg/dose. The no observed adverse effect level (NOAEL) in each study was determined to be 100 mg/kg/dose. A pivotal 6-month chronic dose study in rats is ongoing. Rats are being administered 26 weekly doses of VX15/2503 at 0, 30, 100, or 200 mg/kg/dose; data through 13 weeks have been analyzed. T cell associated SEMA4D remained saturated throughout this phase in mid and high dose animals. On average, high dose rats had a Cmax of 4,378 μg/mL and exposure of 330,878 μgαhr/mL (AUC0-168). Appetance, bodyweight, serum chemistry, behavior, general condition, and ophthalmologic parameters were unaffected by VX15/2503 administration. Treated animals mounted appropriate IgG response; however, low and high dose females produced a slight, but statistically significant lower IgM response than control animals five days following KLH administration. A statistically significant reduction of 33 to 80% was observed in circulating NK cell levels; no similar reduction was observed with macaques dosed weekly X 5 at 100 mg/kg or in ongoing clinical analyses of NK cell levels. Additionally, fewer splenic germinal centers and reduced lymphocyte infiltrates in hepatic parenchyma were observed in the tissues from high-dose animals. These pharmacologic effects did not result in frank toxicity in these animals. A Phase I trial with VX15/2503 is currently being conducted in adult patients with advanced solid tumors. A better understanding of antibody-antigen interactions as well as the nonclinical safety and pharmacology of this antibody will inform clinical development of VX15/2503. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 936. doi:1538-7445.AM2012-936</jats:p

Abstract 4578: Nonclinical safety assessment of VX15/2503: A humanized IgG4 monoclonal antibody to sema4D

Abstract VX15/2503 is a humanized IgG4 monoclonal antibody that binds with approximately 3 nM affinity to Semaphorin 4D (SEMA4D, CD100). SEMA4D is an important mediator of axonal growth cone guidance, vascular development, angiogenesis, and T cell and B cell activation. SEMA4D is found predominantly as a 150 kDa transmembrane protein on lymphocytes and as a 240 kDa, homodimeric, soluble form of the protein. VX15/2503 or its murine progenitor (MAb 67-2) suppressed tumor growth and angiogenesis in syngeneic, xenograft and transgenic tumor models. Toxicology and pharmacology studies of VX15/2503 were performed in Sprague-Dawley rats and cynomolgus macaques using single or five weekly intravenous injections. Toxicology profiles were similar for both species in both studies. Single dose studies employed doses of between 0.01 and 100 mg/kg; repeat dose studies evaluated 10, 30 and 100 mg/kg. No adverse histopathologic or clinical effects were noted in either species injected with doses up to 100 mg/kg. Appetence, body weights, serum chemistry and ophthalmologic factors were also unaffected, as were primate ECG results. Thus the NOAEL was established at 100 mg/kg for both species. Partial to complete T cell associated SEMA4D saturation was observed in all animals across the dose ranges evaluated and SEMA4D saturation was dose dependent. Single dose animals that reached a VX15/2503 serum concentration of ≥2 µg/mL exhibited transient complete T cell saturation. Prolonged saturation occurred at the 100 mg/kg dose level. Repeat dose animals in the 100 mg/kg dose group were on average at least 20% saturated for 134 and 169 days in rats and cynomolgus macaques, respectively. Single dose VX15/2503 half-life values increased with dose and varied from 27 to 246 hours in rats; similar results were obtained from the primate single dose study. Repeat dose Cmax and AUC (exposure) values were higher in both species than those from the single dose studies and steady-state was achieved after the fourth dose. Anti-VX15/2503 responses were detected in the sera of most animals in both studies. Human tissue arrays incubated with VX15/2503 showed diverse distribution of SEMA4D on resident or itinerant lymphocytes in lymphoid tissues as well as in sections of brain, lung and endometrium. Based on these results VX15/2503 was selected for clinical development in oncology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4578. doi:10.1158/1538-7445.AM2011-4578</jats:p

Abstract 5030: Antibody blockade of Semaphorin 4D promotes tumor rejection and improves response to immune checkpoint blockade and chemotherapy

Abstract Semaphorin4D (SEMA4D) normally functions to regulate, adhesion, motility and activation state of multiple cell types including those of the nervous, vascular and immune systems. In the setting of cancer, SEMA4D has been shown to promote tumor progression and metastasis. SEMA4D and its high affinity receptor plexin B1 are broadly expressed in human and murine tumors, and expression levels correlate with invasive disease in several human tumors. The interaction of plexin B1 with MET and ERBB2 can lead to SEMA4D-mediated transactivation of these membrane receptor kinases with a direct effect on tumor cell migration and invasive growth. Moreover, SEMA4D is highly expressed by inflammatory cells that are recruited to the tumor microenvironment. Purpose: Determine the role(s) of SEMA4D in shaping the tumor microenvironment, and investigate the anti-tumor response mediated by antibody neutralization of SEMA4D as a single agent or in combination with other immunomodulatory therapies. Methods: Subcutaneous colon and orthotopic ERBB2+ breast carcinoma models were employed to investigate treatment with a monoclonal antibody to SEMA4D. Anti-tumor response and tumor infiltrating immune cell profiles were characterized by in vivo growth kinetics, immunohistochemistry, FACS, and functional assays. Results: We describe an immunomodulatory function of SEMA4D within the tumor microenvironment, whereby SEMA4D influences the recruitment and activity of cytotoxic CD8+ T cells, the density of B cells within the tumor, and regulates the balance and localization of inflammatory M1 and tolerance-inducing M2 macrophage in tumor stroma. Antibody blockade of SEMA4D modifies the balance of immune cells and production of pro-inflammatory cytokines within the tumor microenvironment, promotes tumor rejection, and induces ∼30% tumor growth delay (TGD) in Colon26 and &amp;gt;70% TGD in Tubo breast tumor models. Furthermore, combination of SEMA4D antibody with immune checkpoint blockade inhibitor antibodies to CTLA4 and PD1, significantly improves frequency of complete tumor rejection (58% and 37%) and increases survival (&amp;gt;295% and &amp;gt;55% TGD respectively) as compared to monotherapy with these targeted agents. Similar combinatorial effects were observed when anti-SEMA4D antibody is administered with immunomodulatory chemotherapy, such as cyclophosphamide, which increases efficacy (81% TGD) and frequency of complete rejection (40%) in tumor engrafted mice. Conclusion: SEMA4D represents a novel immunological target and antibody neutralization of SEMA4D may be a novel therapeutic option for cancer treatment. The humanized anti-SEMA4D antibody, VX15/2503, has successfully completed IND-enabling toxicology testing and the dose escalation phase of a Phase I trial, in which the highest doses were well tolerated in adult patients with advanced solid tumors. Citation Format: Elizabeth E. Evans, Holm Bussler, Sebold Torno, Janaki Veeraraghavan, Alan S. Jonason, Michael A. Doherty, Christine Reilly, Crystal Mallow, Jennifer Seils, Mark Paris, Terrence L. Fisher, William J. Bowers, Maria Scrivens, Leslie Balch, Renee Kirk, Alan Howell, Valerie Iddison, John E. Leonard, Ernest S. Smith, Maurice Zauderer. Antibody blockade of Semaphorin 4D promotes tumor rejection and improves response to immune checkpoint blockade and chemotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5030. doi:10.1158/1538-7445.AM2014-5030</jats:p