Optical measurement of mechanical forces inside short DNA loops

Knowledge of the mechanical properties of double-stranded DNA (dsDNA) is essential to understand the role of dsDNA looping in gene regulation and the mechanochemistry of molecular machines that operate on dsDNA. Here, we use a newly developed tool, force sensors with optical readout, to measure the forces inside short, strained loops composed of both dsDNA and single-stranded DNA. By varying the length of the loops and their proportion of dsDNA, it was possible to vary their internal forces from 1 pN to >20 pN. Surprisingly, internal loop forces changed erratically as the amount of dsDNA was increased for a given loop length, with the effect most notable in the smallest loop (57 nucleotides). Monte Carlo simulations based on the helical wormlike chain model accurately predict internal forces when more than half of the loop is dsDNA but fail otherwise. Mismatches engineered into the double-stranded regions increased flexibility, suggesting that Watson-Crick basepaired dsDNA can withstand high compressive forces without recourse to multibase melts. Fluorescence correlation spectroscopy further excluded transient melting (microsecond to millisecond duration) as a mechanism for relief of compressive forces in the tested dsDNAs. DNA loops with integrated force sensors may allow the comprehensive mapping of the elasticity of short dsDNAs as a function of both sequence and salt.     

Structural and functional determinants in the S5-P region of HCN-encoded pacemaker channels revealed by cysteine-scanning substitutions

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are responsible for the membrane pacemaker current that underlies the spontaneous generation of bioelectrical rhythms. However, their structure-function relationship is poorly understood. Previously, we identified several pore residues that influence HCN gating properties and proposed a pore-to-gate mechanism. Here, we systematically introduced cysteine-scanning substitutions into the descending portion of the P loop (residues 339-345) of HCN1-R (where R is resistance to sulfhydryl-reactive agents) channels, in which all endogenous cysteines except C303 have been removed or replaced. F339C, K340C, A341C, M342C, S343C, and M345C did not produce functional currents. Interestingly, the loss of function phenotype of F339C could be rescued by the reducing agent dithiothreitol (DTT). H344C but not HCN1-R and DTT-treated F339C channels were sensitive to blockade by divalent Cd(2+) (current with 100 microM Cd(2+)/control current at -140 mV = 67.6 +/- 2.9%, 109.3 +/- 3.1%, and 103.8 +/- 1.7%, respectively). Externally applied methanethiosulfate ethylammonium, a covalent sulfhydryl-reactive compound, irreversibly modified H344C by reducing the current at -140 mV (to 43.7 +/- 6.5%), causing a hyperpolarizing steady-state activation shift (change in half-activation voltage: approximately 6 mV) and decelerated gating kinetics (by up to 3-fold). Based on these results, we conclude that pore residues 339-345 are important determinants of the structure-function properties of HCN channels and that the side chain of H344 is externally accessible.     

Myogenin and oxidative enzyme gene expression levels are elevated in rat soleus muscles after endurance training.

The intent of this study was to determine whether endurance exercise training regulates increases in metabolic enzymes, which parallel modulations of myogenin and MyoD in skeletal muscle of rats. Adult Sprague-Dawley rats were endurance trained (TR) 5 days weekly for 8 wk on a motorized treadmill. They were killed 48 h after their last bout of exercise. Sedentary control (Con) rats were killed at the same time as TR animals. Myogenin, MyoD, citrate synthase (CS), cytochrome-c oxidase (COX) subunits II and VI, lactate dehydrogenase (LDH), and myosin light chain mRNA contents were determined in soleus muscles by using RT-PCR. Myogenin mRNA content was also estimated by using dot-blot hybridization. Protein expression levels of myogenin and MyoD were measured by Western blots. CS enzymatic activity was also measured. RT-PCR measurements showed that the mRNA contents of myogenin, CS, COX II, COX VI, and LDH were 25, 20, 17, 16, and 18% greater, respectively, in TR animals compared with Con animals (P < 0.05). The ratio of myogenin to MyoD mRNA content estimated by RT-PCR in TR animals was 28% higher than that in Con animals (P < 0.05). Myosin light chain expression was similar in Con and TR muscles. Results from dot-blot hybridization to a riboprobe further confirmed the increase in myogenin mRNA level in TR group. Western blot analysis indicated a 24% greater level of myogenin protein in TR animals compared with Con animals (P < 0.01). The soleus muscles from TR animals had a 25% greater CS enzymatic activity than the Con animals (P < 0.01). Moreover, myogenin mRNA and protein contents were positively correlated to CS activity and mRNA contents of CS, COX II, and COX VI (P < 0.05). These data are consistent with the hypothesis that myogenin is in the pathway for exercise-induced changes in mitochondrial enzymes.     

Soluble ACE2-mediated cell entry of SARS-CoV-2 via interaction with proteins related to the renin-angiotensin system

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Soy‐Derived Phytochemical Genistein Modifies Chromatome Topology to Restrict Cancer Cell Proliferation

Epidemiological data indicate that human cancer risk is significantly reduced by the consumption of soy‐based foods containing the “phytoestrogen” genistein, which can signal via host cell estrogen receptors. While additional chemoprotective effects of genistein induced by epigenetic factors have also been reported, the key molecules and mechanisms involved are poorly defined. We therefore investigated genistein effects on chromatin‐bound proteins in the estrogen receptor‐deficient cell line MDA‐MB‐231 which is insensitive to phytoestrogen signaling. After exposure to low‐dose genistein for >1 month, MDA‐MB‐231 cells exhibited stable epigenetic alterations that are analyzed via partial MNase digestion and TMT‐based quantitative proteomics. 3177 chromatin‐bound proteins are identified with high confidence, including 882 molecules that displayed altered binding topology after cell conditioning with genistein. Prolonged phytochemical exposure conferred heritable changes in the binding topology of key epigenetic regulators including ATRX, SUV39H1/H2, and HP1BP3 that are preserved in untreated progeny, resulting in sustained downregulation of proliferation genes and reduced cell growth. These data indicate that soy derivative genistein exerts complex estrogen receptor‐independent effects on the epigenome likely to influence tumorigenesis by restricting cell growth.     

Present population and habitat status of potentially threatened Asian horseshoe crabs <Emphasis Type="Italic">Tachypleus tridentatus</Emphasis> and <Emphasis Type="Italic">Carcinoscorpius rotundicauda</Emphasis> in Hong Kong: a proposal for marine protected areas

While there are persistent calls for developing more marine protected areas (MPAs) for Asian horseshoe crab conservation in response to population declines in Asia–Pacific region, most existing horseshoe crab MPAs were designated without prior comprehensive population assessment and habitat characterization. This study collected biological and habitat information in Hong Kong, in order to identify priority sites for conservation and management of Asian horseshoe crab populations. The territory-wide surveys at 18 spawning/nursery beaches displayed a persistently low mean juvenile density from 0.16 to 2.19 ind/100 m² in 2012 and 0.08 to 1.41 ind/100 m² in 2014. These density data were within the same range of that in 2002 and 2007 (0.10–1.97 ind/100 m²), apart from a low survey return of 0.08–0.31 ind/100 m² in 2004. The current population of juvenile T. tridentatus and C. rotundicauda in Hong Kong is estimated about 2100–4300 and 2400–3000 individuals, respectively. From the 2012–2014 data, no new recruitments of 1st–3rd instar juveniles were found, as the shores were occupied mostly by 6th–9th instar juveniles of prosomal width between 23 and 45 mm, in which considerably high mortality rates were noted. The present findings of existing small and discrete juvenile populations, coupled with relatively few recruits, suggest that the status of juvenile horseshoe crabs in Hong Kong is fragile and vulnerable to local extirpation, especially if no urgent protection measures are implemented. Based on available population data and habitat characteristics, three MPAs, ranging from 5 to 7 km², are proposed, so as to conserve over 60 and 70 % of the existing juvenile populations of T. tridentatus and C. rotundicauda, respectively. The approach adopted in this study may serve as a case study for proposing other horseshoe crab MPAs in Asia, where increasing human disturbances and over-harvest are imminent.     

Cloning, tissue distribution, subcellular localization and overexpression of murine histidine-rich Ca2+ binding protein.

The histidine-rich Ca2+ binding protein (HRC) resides in the sarcoplasmic reticulum of muscle and binds Ca2+. Since Ca2+ concentrations can regulate gene expression via calcineurin, the mouse homologue of HRC (mHRC) was isolated and characterized. mHRC was detected in muscle progenitor cells, in primary clonal thymic tumors and a tumor cell line, suggesting a broader role for mHRC than in Ca2+ storage during muscle contraction. mHRC was present in the perinuclear region of myoblasts. To examine if it can regulate gene expression, mHRC was overexpressed in cells differentiating into cardiac and skeletal muscle. mHRC had no effect on cardiogenesis or myogenesis. Therefore, if mHRC plays a role in the regulation of gene expression during cellular differentiation, it does not appear to be either rate-limiting or inhibitory.     

The legumain McPAL1 from Momordica cochinchinensis is a highly stable Asx-specific splicing enzyme

Legumains, also known as asparaginyl endopeptidases (AEPs), cleave peptide bonds after Asn/Asp (Asx) residues. In plants, certain legumains also have ligase activity that catalyzes biosynthesis of Asx-containing cyclic peptides. An example is the biosynthesis of MCoTI-I/II, a squash family-derived cyclic trypsin inhibitor, which involves splicing to remove the N-terminal prodomain and then N-to-C-terminal cyclization of the mature domain. To identify plant legumains responsible for the maturation of these cyclic peptides, we have isolated and characterized a legumain involved in splicing, McPAL1, from Momordica cochinchinensis (Cucurbitaceae) seeds. Functional studies show that recombinantly expressed McPAL1 displays a pH-dependent, trimodal enzymatic profile. At pH 4 to 6, McPAL1 selectively catalyzed Asp-ligation and Asn-cleavage, but at pH 6.5 to 8, Asn-ligation predominated. With peptide substrates containing N-terminal Asn and C-terminal Asp, such as is found in precursors of MCoTI-I/II, McPAL1 mediates proteolysis at the Asn site and then ligation at the Asp site at pH 5 to 6. Also, McPAL1 is an unusually stable legumain that is tolerant of heat and high pH. Together, our results support that McPAL1 is a splicing legumain at acidic pH that can mediate biosynthesis of MCoTI-I/II. We purport that the high thermal and pH stability of McPAL1 could have applications for protein engineering.