In vitro thermal stress induces apoptosis and reduces development of porcine parthenotes

The precise physiological causes that result in reduced development of oocytes after heat shock (HS) are not clear. In this study, apoptosis, heat shock protein70 (hsp70), and in vitro development of porcine oocytes were evaluated after HS. Porcine cumulus-oocyte complexes (COCs) were subjected to in vitro maturation for 42 h. The matured oocytes were then heated at 41.5 degrees C for 0 h (control, C0h), 1 h (HS1h), 2 h (HS2h), or 4 h (HS4h). An additional group of oocytes was cultured for 4 h without HS (control, C4h). In Experiment 1, expression of hsp70 was detected by Western-blotting and no difference between controls and HS groups was observed. In Experiment 2, apoptosis of matured oocytes after HS was examined by Annexin V-FITC and TUNEL. No significant TUNEL-positive signals were detected in the heated oocytes compared to the controls, but the intensity of Annexin V-FITC labeling among different groups increased with length of HS and in vitro culture (P<0.05). Oocytes were parthenogenetically activated by an electric pulse plus 6-DMAP (Experiment 3). Mean (+/-S.E.M.) embryonic development in HS2h (cleavage: 42+/-29%; blastocyst: 11+/-10%) and HS4h (cleavage: 36+/-28%; blastocyst: 11+/-8%) were decreased when compared to those in C0h (cleavage: 63+/-12%; blastocyst: 24+/-14%) and C4h (cleavage: 66+/-8%; blastocyst: 21+/-11%). Numbers of blastocysts with TUNEL-positive signals were similar among groups, but the signals increased before the eight-cell stage in HS groups (P<0.05). In conclusion, developmental competence of matured pig oocytes was compromised after heat shock, but it was not closely associated with the expression of oocyte hsp70. However, there may be a link between apoptosis and developmental competence of porcine oocytes.     

Oxygen- and growth rate-dependent regulation of Escherichia coli fumarase (FumA, FumB, and FumC) activity

Escherichia coli contains three biochemically distinct fumarases which catalyze the interconversion of fumarate to L-malate in the tricarboxylic acid cycle. Batch culture studies indicated that fumarase activities varied according to carbon substrate and cell doubling time. Growth rate control of fumarase activities in the wild type and mutants was demonstrated in continuous culture; FumA and FumC activities were induced four- to fivefold when the cell growth rate (k) was lowered from 1.2/h to 0.24/h at 1 and 21% O(2), respectively. There was a twofold induction of FumA and FumC activities when acetate was utilized instead of glucose as the sole carbon source. However, these fumarase activities were still shown to be under growth rate control. Thus, the activity of the fumarases is regulated by the cell growth rate and carbon source utilization independently. Further examination of FumA and FumC activities in a cya mutant suggested that growth rate control of FumA and FumC activities is cyclic AMP dependent. Although the total fumarase activity increased under aerobic conditions, the individual fumarase activities varied under different oxygen levels. While FumB activity was maximal during anaerobic growth (k = 0.6/h), FumA was the major enzyme under anaerobic cell growth, and the maximum activity was achieved when oxygen was elevated to 1 to 2%. Further increase in the oxygen level caused inactivation of FumA and FumB activities by the high oxidized state, but FumC activity increased simultaneously when the oxygen level was higher than 4%. The same regulation of the activities of fumarases in response to different oxygen levels was also found in mutants. Therefore, synthesis of the three fumarase enzymes is controlled in a hierarchical fashion depending on the environmental oxygen that the cell encounters.     

Identifying Primate ACE2 Variants That Confer Resistance to SARS-CoV-2

SARS-CoV-2 infects humans through the binding of viral S-protein (spike protein) to human angiotensin I converting enzyme 2 (ACE2). The structure of the ACE2-S-protein complex has been deciphered and we focused on the 27 ACE2 residues that bind to S-protein. From human sequence databases, we identified nine ACE2 variants at ACE2–S-protein binding sites. We used both experimental assays and protein structure analysis to evaluate the effect of each variant on the binding affinity of ACE2 to S-protein. We found one variant causing complete binding disruption, two and three variants, respectively, strongly and mildly reducing the binding affinity, and two variants strongly enhancing the binding affinity. We then collected the ACE2 gene sequences from 57 nonhuman primates. Among the 6 apes and 20 Old World monkeys (OWMs) studied, we found no new variants. In contrast, all 11 New World monkeys (NWMs) studied share four variants each causing a strong reduction in binding affinity, the Philippine tarsier also possesses three such variants, and 18 of the 19 prosimian species studied share one variant causing a strong reduction in binding affinity. Moreover, one OWM and three prosimian variants increased binding affinity by >50%. Based on these findings, we proposed that the common ancestor of primates was strongly resistant to and that of NWMs was completely resistant to SARS-CoV-2 and so is the Philippine tarsier, whereas apes and OWMs, like most humans, are susceptible. This study increases our understanding of the differences in susceptibility to SARS-CoV-2 infection among primates.     

Par-4 links dopamine signaling and depression

Prostate apoptosis response 4 (Par-4) is a leucine zipper containing protein that plays a role in apoptosis. Although Par-4 is expressed in neurons, its physiological role in the nervous system is unknown. Here we identify Par-4 as a regulatory component in dopamine signaling. Par-4 directly interacts with the dopamine D2 receptor (D2DR) via the calmodulin binding motif in the third cytoplasmic loop. Calmodulin can effectively compete with Par-4 binding in a Ca2+-dependent manner, providing a route for Ca2+-mediated downregulation of D2DR efficacy. To examine the importance of the Par-4/D2DR interaction in dopamine signaling in vivo, we used a mutant mouse lacking the D2DR interaction domain of Par-4, Par-4DeltaLZ. Primary neurons from Par-4DeltaLZ embryos exhibit an enhanced dopamine-cAMP-CREB signaling pathway, indicating an impairment in dopamine signaling in these cells. Remarkably, Par-4DeltaLZ mice display significantly increased depression-like behaviors. Collectively, these results provide evidence that Par-4 constitutes a molecular link between impaired dopamine signaling and depression.     

Removal of high concentration of NH3 and coexistent H2S by biological activated carbon (BAC) biotrickling filter

High efficiency of NH3 and H2S removal from waste gases was achieved by the biotrickling filter. Granular activated carbon (GAC), inoculated with Arthrobacter oxydans CH8 for NH3 removal and Pseudomonas putida CH11 for H2S removal, was used as packing material. Under conditions in which 100% H2S was removed, extensive tests to eliminate high concentrations of NH3 emission-including removal characteristics, removal efficiency, and removal capacity of the system-were performed. The results of the Bed Depth Service Time (BDST) experiment suggested that physical adsorption of NH3 gas by GAC was responsible for the first 10 days, after which NH3 gas was biodegraded by inoculated microorganisms. The dynamic steady state between physical adsorption and biodegradation was about two weeks. After the system achieved equilibrium, the BAC biotrickling filter exhibited high adaptation to shock loading, elevated temperature, and flow rate. Greater than 96% removal efficiency for NH3 was achieved during the 140-day operating period when inlet H2S loading was maintained at 6.25 g-S/m3/h. During the operating period, the pH varied between 6.5 and 8.0 after the physical adsorption stage, and no acidification or alkalinity was observed. The results also demonstrated that NH3 removal was not affected by the coexistence of H2S while gas retention time was the key factor in system performance. The retention time of at least 65 s is required to obtain a greater than 95% NH3 removal efficiency. The critical loading of NH3 for the system was 4.2 g-N/m3/h, and the maximal loading was 16.2 g-N/m3/h. The results of this study could be used as a guide for further design and operation of industrial-scale systems.     

Biodegradation of octylphenol polyethoxylate surfactant Triton X-100 by selected microorganisms

Octylphenol polyethoxylate (OPEO(n)) surfactants are used in numerous commercial and industrial products. Large amounts of such surfactants and their various residual biodegradation by-products are ultimately released into the environment. OPEO(n) biodegradation was performed in this study using pure cultures of Pseudomonas species and strains under different environmental conditions. Environmental factors including the pH, nitrogen sources, and growth kinetics of the cells were investigated. The intermediates of Triton X-100 biotransformation were detected by high performance liquid chromatography-mass spectrophotograph (HPLC-MS). We found the highest specific growth rate (mu) was 0.56 h(-1) and this was achieved by strain E with an initial concentration of Triton X-100 of 5000 mg L(-1). A pH level of 7 was most favorable for cell growth for all five strains. The highest specific growth rate was achieved using (NH(4))(2)SO(4) as the sole nitrogen source for strain E. Strain A showed an enhancement of growth when between 0.2 and 1.4 mg L(-1) of H(2)O(2) was added. Detection of intermediates was possible after four days of transformation and the octylphenol triethoxylate (OPEO(3)) peak was predominant, while the high molecular weight peaks had all disappeared. The kinetic analysis demonstrated that the greatest maximum specific growth rate (mu(max)) and the greatest saturation constant (K(s)) of 0.83 h(-1) and 5.24 mg L(-1), respectively, were obtained for strain E in 5000 mg L(-1) Triton X-100. The higher K(i) revealed that strain A was resistant to higher Triton X-100 concentrations.     

How actin crosslinking and bundling proteins cooperate to generate an enhanced cell mechanical response

Actin-crosslinking proteins organize actin filaments into dynamic and complex subcellular scaffolds that orchestrate important mechanical functions, including cell motility and adhesion. Recent mutation studies have shown that individual crosslinking proteins often play seemingly non-essential roles, leading to the hypothesis that they have considerable redundancy in function. We report live-cell, in vitro, and theoretical studies testing the mechanical role of the two ubiquitous actin-crosslinking proteins, alpha-actinin and fascin, which co-localize to stress fibers and the basis of filopodia. Using live-cell particle tracking microrheology, we show that the addition of alpha-actinin and fascin elicits a cell mechanical response that is significantly greater than that originated by alpha-actinin or fascin alone. These live-cell measurements are supported by quantitative rheological measurements with reconstituted actin filament networks containing pure proteins that show that alpha-actinin and fascin can work in concert to generate enhanced cell stiffness. Computational simulations using finite element modeling qualitatively reproduce and explain the functional synergy of alpha-actinin and fascin. These findings highlight the cooperative activity of fascin and alpha-actinin and provide a strong rationale that an evolutionary advantage might be conferred by the cooperative action of multiple actin-crosslinking proteins with overlapping but non-identical biochemical properties. Thus the combination of structural proteins with similar function can provide the cell with unique properties that are required for biologically optimal responses.