Protein targets for structure-based anti-Mycobacterium tuberculosis drug discovery

Mycobacterium tuberculosis, which belongs to the genus Mycobacterium, is the pathogenic agent for most tuberculosis (TB). As TB remains one of the most rampant infectious diseases, causing morbidity and death with emergence of multi-drug-resistant and extensively-drug-resistant forms, it is urgent to identify new drugs with novel targets to ensure future therapeutic success. In this regards, the structural genomics of M. tuberculosis provides important information to identify potential targets, perform biochemical assays, determine crystal structures in complex with potential inhibitor(s), reveal the key sites/residues for biological activity, and thus validate drug targets and discover novel drugs. In this review, we will discuss the recent progress on novel targets for structure-based anti-M. tuberculosis drug discovery.     

Mating system and pollination biology of a high-mountain perennial plant,Rhodiola dumulosa (Crassulaceae)

Aims Rhodiola dumulosa is a perennial herb growing in a naturally fragmented habitat of high-mountain rocks. This research aims to (i) investigate the mating system characteristics and pollination biology of R. dumulosa, (ii) study the effects of ecological factors on the mating system and pollination biology of R. dumulosa and (iii) assess the relationship between its pollination and mating system.Methods Mating system parameters were analysed using allozyme markers. Growth of pollen tubes from artificial self- and cross-pollination was also examined. Field investigations were conducted on insect flower visitation during the blooming period of R. dumulosa. Relationships among mating system, insect flower visitation frequency and environmental factors were assessed.Important findings The results showed that (i) R. dumulosa has a mixed-mating system: it is self-compatible and primarily outbreeding, with the multilocus outcrossing rate (t m) ranging from 0.589 ± 0.078 (± standard deviation) to 0.846 ± 0.077; inbreeding coefficients of maternal parents (F) were zero, indicating that inbreeding depression was intense. (ii) Light intensity and temperature impacted the visitation of the principal pollinators, the syrphid Eristalis tenax and the bumblebee Bombus pyrosoma. (iii) Population size plays an important role in the mating system and pollination. Populations received more frequent visits by the principal pollinators usually had higher outcrossing rates. Pollination to some extent affects the mating system of R. dumulosa and it is important to maintain large populations to prevent further inbreeding and to maintain pollinators to facilitate outcrossing.     

GmZIP1 encodes a symbiosis-specific zinc transporter in soybean.

The importance of zinc in organisms is clearly established, and mechanisms involved in zinc acquisition by plants have recently received increased interest. In this report, the identification, characterization and location of GmZIP1, the first soybean member of the ZIP family of metal transporters, are described. GmZIP1 was found to possess eight putative transmembrane domains together with a histidine-rich extra-membrane loop. By functional complementation of zrt1zrt2 yeast cells no longer able to take up zinc, GmZIP1 was found to be highly selective for zinc, with an estimated K(m) value of 13.8 microm. Cadmium was the only other metal tested able to inhibit zinc uptake in yeast. An antibody raised against GmZIP1 specifically localized the protein to the peribacteroid membrane, an endosymbiotic membrane in nodules resulting from the interaction of the plant with its microsymbiont. The specific expression of GmZIP1 in nodules was confirmed by Northern blot, with no expression in roots, stems, or leaves of nodulated soybean plants. Antibodies to GmZIP1 inhibited zinc uptake by symbiosomes, indicating that at least some of the zinc uptake observed in isolated symbiosomes could be attributed to GmZIP1. The orientation of the protein in the membrane and its possible role in the symbiosis are discussed.     

Mechanical activation of spike fosters SARS-CoV-2 viral infection

The outbreak of SARS-CoV-2 (SARS2) has caused a global COVID-19 pandemic. The spike protein of SARS2 (SARS2-S) recognizes host receptors, including ACE2, to initiate viral entry in a complex biomechanical environment. Here, we reveal that tensile force, generated by bending of the host cell membrane, strengthens spike recognition of ACE2 and accelerates the detachment of spike’s S1 subunit from the S2 subunit to rapidly prime the viral fusion machinery. Mechanistically, such mechano-activation is fulfilled by force-induced opening and rotation of spike’s receptor-binding domain to prolong the bond lifetime of spike/ACE2 binding, up to 4 times longer than that of SARS-S binding with ACE2 under 10 pN force application, and subsequently by force-accelerated S1/S2 detachment which is up to ~10³ times faster than that in the no-force condition. Interestingly, the SARS2-S D614G mutant, a more infectious variant, shows 3-time stronger force-dependent ACE2 binding and 35-time faster force-induced S1/S2 detachment. We also reveal that an anti-S1/S2 non-RBD-blocking antibody that was derived from convalescent COVID-19 patients with potent neutralizing capability can reduce S1/S2 detachment by 3 × 10⁶ times under force. Our study sheds light on the mechano-chemistry of spike activation and on developing a non-RBD-blocking but S1/S2-locking therapeutic strategy to prevent SARS2 invasion.Subject terms: Molecular biology, Structural biology     

Host-recognition kairomone from Sogatella furcifera for the parasitoid Anagrus nilaparvatae

A kairomone produced by the rice white-backed planthopper Sogatella furcifera (Horvath) for the mymarid egg parasitoid Anagrus nilaparvatae Pang et Wang was investigated. Eggs, female and male adults, nymphs, exuvia, honeydew, nymph-damaged plants and plants with S. furcifera eggs all elicited searching behavior in the parasitoid. Eggs, female adults and plants with eggs were the most attractive, while exuvia and honeydew evoked the weakest responses. The active compound(s) from S. furcifera female adults, nymphs and plants with eggs could be effectively extracted with methanol, acetone, n-hexane, ethyl ether and dichloromethane. Isolation of the active chemical(s) in three solvent extracts, the acetone and n-hexane extracts of S. furcifera female adults, and the acetone extract of S. furcifera nymphs, was accomplished by thin-layer chromatography. Only one fraction was active and had the same chemical properties in the three solvent extracts. Infra-red analysis of this fraction revealed a kind of ester with saturated and unsaturated fatty acids, which was previously found to be active in the same compound as the extracts from the nymphs and female adults of the rice brown planthopper, Nilaparvata lugens (Stål), and identified as palm oil. The results are discussed in relation to host location by A. nilaparvatae.     

Polypyrimidine Tract-Binding Protein Positively Regulates Inclusion of an Alternative 3′-Terminal Exon

Polypyrimidine tract-binding protein (PTB) is an abundant vertebrate hnRNP protein. PTB binding sites have been found within introns both upstream and downstream of alternative exons in a number of genes that are negatively controlled by the binding of PTB. We have previously reported that PTB binds to a pyrimidine tract within an RNA processing enhancer located adjacent to an alternative 3′-terminal exon within the gene coding for calcitonin and calcitonin gene-related peptide. The enhancer consists of a pyrimidine tract and CAG directly abutting on a 5′ splice site sequence to form a pseudoexon. Here we show that the binding of PTB to the enhancer pyrimidine tract is functional in that exon inclusion increases when in vivo levels of PTB increase. This is the first example of positive regulation of exon inclusion by PTB. The binding of PTB was antagonistic to the binding of U2AF to the enhancer-located pyrimidine tract. Altering the enhancer pyrimidine tract to a consensus sequence for the binding of U2AF eliminated enhancement of exon inclusion in vivo and exon polyadenylation in vitro. An additional PTB binding site was identified close to the AAUAAA hexanucleotide sequence of the exon 4 poly(A) site. These observations suggest a dual role for PTB in facilitating recognition of exon 4: binding to the enhancer pyrimidine tract to interrupt productive recognition of the enhancer pseudoexon by splicing factors and interacting with the poly(A) site to positively affect polyadenylation.     

An EGP-2/Ep-CAM-expressing transgenic rat model to evaluate antibody-mediated immunotherapy

The human pancarcinoma-associated epithelial glycoprotein-2 (EGP-2), also known as 17-1A or Ep-CAM, is a 38-kDa transmembrane antigen, commonly used for targeted immunotherapy of carcinomas. Although strongly expressed by most carcinomas, EGP-2 is also expressed in most simple epithelia. To evaluate treatment-associated effects and side-effects on tumor and normal tissue respectively, we generated an EGP-2-expressing transgenic Wistar rat. To express the cDNA of the EGP-2 in an epithelium-specific manner, the 5′ and 3′ distal flanking regions of the human keratin 18 (K18) gene were used. EGP-2 protein expression was observed in the liver and pancreas, whereas EGP-2 mRNA could also be detected in lung, intestine, stomach and kidney tissues. In this rat, EGP-2-positive tumors can be induced by injecting a rat-derived carcinoma cell line transfected with the GA733-2 cDNA encoding EGP-2. Transgenic rats were used to study specific in vivo localization of an i.v. anti-EGP-2 monoclonal antibody, MOC31, applied i.v. Immunohistochemical analyses showed the specific localization of MOC31 in s.c. induced EGP-2-positive tumors, as well as in the liver. In contrast, in EGP-2-transgenic rats, MOC31 did not bind to EGP-2-negative tumors, the pancreas, or other normal tissues in vivo. In conclusion, an EGP-2-transgenic rat model has been generated that serves as a model to evaluate the efficacy and safety of a variety of anti-EGP-2-based immunotherapeutic modalities. Received: 9 March 1999 / Accepted: 6 May 1999     

Isolation and Identification of Steroidal Sapogenins from Rhizome of Dioscorea septemloba Thunb

Five components were isolated from acid-treated rhizome of D. septemloba Thunb. Their structures, the were identified as diosgenin (Ⅰ), β-sitosterol (Ⅱ), palmitic acid (Ⅲ), diosgenin palmitate (Ⅳ) and △3,5-deoxytigogenin (Ⅴ) on the basis of physical constants and spectral data.