Exceptional Simian Hemorrhagic Fever Virus Diversity in a Wild African Primate Community

Simian hemorrhagic fever virus (SHFV) is an arterivirus that causes severe disease in captive macaques. We describe two new SHFV variants subclinically infecting wild African red-tailed guenons (Cercopithecus ascanius). Both variants are highly divergent from the prototype virus and variants infecting sympatric red colobus (Procolobus rufomitratus). All known SHFV variants are monophyletic and share three open reading frames not present in other arteriviruses. Our data suggest a need to modify the current arterivirus classification.     

Overexpression of Bcl(XL) in B cells promotes Th1 response and exacerbates collagen-induced arthritis

B cells play a pathogenic or regulatory role in many autoimmune diseases through production of autoantibodies, cytokine production, and Ag presentation. However, the mechanisms that regulate these B cell functions under different autoimmune settings remain unclear. In the current study, we found that when B cells overexpress an antiapoptotic gene, Bcl(XL), they significantly increased production of IFN-gamma and enhanced Th1 response. Consistently, Bcl-x(L) transgenic mice developed more severe and sustained collagen-induced arthritis due to the enhanced Th1 response. The production of autoantibodies in Bcl(XL) transgenic mice was comparable to that in wild-type mice. Thus, our results indicate a novel role of Bcl(XL) in regulating B cell functions and immune responses. In patients with rheumatoid arthritis, arthritogenic B cells often up-regulate Bcl(XL) expression, which may not only render B cells resistant to apoptosis but also alter the ability of the autoreactive B cells to produce cytokines and modulate the inflammatory response. This may have therapeutic implications if Bcl(XL) expression can be down-regulated in autoreactive B cells.     

Nitric Oxide Synthase and Breast Cancer: Role of TIMP-1 in NO-mediated Akt Activation

Prediction of therapeutic response and cancer patient survival can be improved by the identification of molecular markers including tumor Akt status. A direct correlation between NOS2 expression and elevated Akt phosphorylation status has been observed in breast tumors. Tissue inhibitor matrix metalloproteinase-1 (TIMP-1) has been proposed to exert oncogenic properties through CD63 cell surface receptor pathway initiation of pro-survival PI3k/Akt signaling. We employed immunohistochemistry to examine the influence of TIMP-1 on the functional relationship between NOS2 and phosphorylated Akt in breast tumors and found that NOS2-associated Akt phosphorylation was significantly increased in tumors expressing high TIMP-1, indicating that TIMP-1 may further enhance NO-induced Akt pathway activation. Moreover, TIMP-1 silencing by antisense technology blocked NO-induced PI3k/Akt/BAD phosphorylation in cultured MDA-MB-231 human breast cancer cells. TIMP-1 protein nitration and TIMP-1/CD63 co-immunoprecipitation was observed at NO concentrations that induced PI3k/Akt/BAD pro-survival signaling. In the survival analysis, elevated tumor TIMP-1 predicted poor patient survival. This association appears to be mainly restricted to tumors with high NOS2 protein. In contrast, TIMP-1 did not predict poor survival in patient tumors with low NOS2 expression. In summary, our findings suggest that tumors with high TIMP-1 and NOS2 behave more aggressively by mechanisms that favor Akt pathway activation.     

Weak partitioning chromatography for anion exchange purification of monoclonal antibodies

Weak partitioning chromatography (WPC) is an isocratic chromatographic protein separation method performed under mobile phase conditions where a significant amount of the product protein binds to the resin, well in excess of typical flowthrough operations. The more stringent load and wash conditions lead to improved removal of more tightly binding impurities, although at the cost of a reduction in step yield. The step yield can be restored by extending the column load and incorporating a short wash at the end of the load stage. The use of WPC with anion exchange resins enables a two-column cGMP purification platform to be used for many different mAbs. The operating window for WPC can be easily established using high throughput batch-binding screens. Under conditions that favor very strong product binding, competitive effects from product binding can give rise to a reduction in column loading capacity. Robust performance of WPC anion exchange chromatography has been demonstrated in multiple cGMP mAb purification processes. Excellent clearance of host cell proteins, leached Protein A, DNA, high molecular weight species, and model virus has been achieved.     

High-throughput screening of chromatographic separations: IV. Ion-exchange

Ion-exchange (IEX) chromatography steps are widely applied in protein purification processes because of their high capacity, selectivity, robust operation, and well-understood principles. Optimization of IEX steps typically involves resin screening and selection of the pH and counterion concentrations of the load, wash, and elution steps. Time and material constraints associated with operating laboratory columns often preclude evaluating more than 20-50 conditions during early stages of process development. To overcome this limitation, a high-throughput screening (HTS) system employing a robotic liquid handling system and 96-well filterplates was used to evaluate various operating conditions for IEX steps for monoclonal antibody (mAb) purification. A screening study for an adsorptive cation-exchange step evaluated eight different resins. Sodium chloride concentrations defining the operating boundaries of product binding and elution were established at four different pH levels for each resin. Adsorption isotherms were measured for 24 different pH and salt combinations for a single resin. An anion-exchange flowthrough step was then examined, generating data on mAb adsorption for 48 different combinations of pH and counterion concentration for three different resins. The mAb partition coefficients were calculated and used to estimate the characteristic charge of the resin-protein interaction. Host cell protein and residual Protein A impurity levels were also measured, providing information on selectivity within this operating window. The HTS system shows promise for accelerating process development of IEX steps, enabling rapid acquisition of large datasets addressing the performance of the chromatography step under many different operating conditions.     

Effects of Environmental and Social Conditions on Homosexual Pairing in the Japanese Beetle (Popillia japonica Newman)

Homosexual pairing between males occurs under natural conditions in a wide variety of taxa, including many insect species, but few studies have investigated how environmental and social conditions affect same-sex pairing in insects. We investigated factors affecting homosexual pairing in male Japanese beetles (Popillia japonica Newman) in the field and in the laboratory. Specifically, we investigated how time of day, sex ratio, beetle density, and temperature affected the likelihood of homosexual pairing. In the field, male–male pairs constituted 1–6% of the pairs we collected. Homosexual pairs were more common in the afternoon than in the morning and the evening. Sex ratio, density, and temperature were all related to the likelihood of finding a homosexual pair, but the relationships were not linear. In the laboratory, higher male densities and relatively male-biased sex ratios were associated with an increase in the frequency of homosexual pairs. Homosexual pairs were more frequent at relatively low and relatively high temperatures. Males that mounted other males tended to be smaller than the males that they mounted. In addition, compared to males that were not homosexually paired, there was some indication that the mounting males were smaller, and the mounted males larger, than the unpaired males. Our data suggest that homosexual pairs are a result of males mistaking other males for females, and we hypothesize that the environmental and social factors cause changes in homosexual pairing through their effects on the frequency of pair formation and pair duration.     

Acetate Synthesis from H2 plus CO2 by Termite Gut Microbes

Gut microbiota from Reticulitermes flavipes termites catalyzed an H₂-dependent total synthesis of acetate from CO₂. Rates of H₂-CO₂ acetogenesis in vitro were 1.11 ± 0.37 μmol of acetate g (fresh weight)⁻¹ h⁻¹ (equivalent to 4.44 ± 1.47 nmol termite⁻¹ h⁻¹) and could account for approximately 1/3 of all the acetate produced during the hindgut fermentation. Formate was also produced from H₂ + CO₂, as were small amounts of propionate, butyrate, and lactate-succinate. However, H₂-CO₂ formicogenesis seemed largely unrelated to acetogenesis and was believed not to be a significant reaction in situ. Little or no CH₄ was formed from H₂ + CO₂ or from acetate. H₂-CO₂ acetogenesis was inhibited by O₂, KCN, CHCl₃, and iodopropane and could be abolished by prefeeding R. flavipes with antibacterial drugs. By contrast, prefeeding R. flavipes with starch resulted in almost complete defaunation but had little effect on H₂-CO₂ acetogenesis, suggesting that bacteria were the acetogenic agents in the gut. H₂-CO₂ acetogenesis was also observed with gut microbiota from Prorhinotermes simplex, Zootermopsis angusticollis, Nasutitermes costalis, and N. nigriceps; from the wood-eating cockroach Cryptocercus punctulatus; and from the American cockroach Periplaneta americana. Pure cultures of H₂-CO₂-acetogenic bacteria were isolated from N. nigriceps, and a preliminary account of their morphological and physiological properties is presented. Results indicate that in termites, CO₂ reduction to acetate, rather than to CH₄, represents the main electron sink reaction of the hindgut fermentation and can provide the insects with a significant fraction (ca. 1/3) of their principal oxidizable energy source, acetate.