The FMRP/GRK4 mRNA interaction uncovers a new mode of binding of the Fragile X mental retardation protein in cerebellum

Fragile X syndrome (FXS), the most common form of inherited intellectual disability, is caused by the silencing of the FMR1 gene encoding an RNA-binding protein (FMRP) mainly involved in translational control. We characterized the interaction between FMRP and the mRNA of GRK4, a member of the guanine nucleotide-binding protein (G protein)-coupled receptor kinase super-family, both in vitro and in vivo. While the mRNA level of GRK4 is unchanged in the absence or in the presence of FMRP in different regions of the brain, GRK4 protein level is increased in Fmr1-null cerebellum, suggesting that FMRP negatively modulates the expression of GRK4 at the translational level in this brain region. The C-terminal region of FMRP interacts with a domain of GRK4 mRNA, that we called G4RIF, that is folded in four stem loops. The SL1 stem loop of G4RIF is protected by FMRP and is part of the S1/S2 sub-domain that directs translation repression of a reporter mRNA by FMRP. These data confirm the role of the G4RIF/FMRP complex in translational regulation. Considering the role of GRK4 in GABAB receptors desensitization, our results suggest that an increased GRK4 levels in FXS might contribute to cerebellum-dependent phenotypes through a deregulated desensitization of GABAB receptors.     

Rapid test for determination of urea hydrolysis

A urease test for the rapid determination of urea hydrolysis is described in which diluted urea agar concentrate was used in small amounts with dense inoculum of the test organisms. The method was evaluated and compared with Christensen's urea agar slants by using 728 clinical isolates of gram-negative bacteria. Of the 325 strains of urease-positive Proteus-Providencia-Morganella, 282 (87%) gave positive results within 5 min with the rapid test. Urease activity of 97% of these organisms became evident within 30 min. All 287 isolates which showed no urease activity on Christensen's urea agar also remained negative by this test.     

Additivity-based design of the strongest possible turkey ovomucoid third domain inhibitors for porcine pancreatic elastase (PPE) and Streptomyces griseus protease B (SGPB)

We describe here successful designs of strong inhibitors for porcine pancreatic elastase (PPE) and Streptomyces griseus protease B (SGPB). For each enzyme two inhibitor variants were designed. In one, the reactive site residue (position 18) was retained and the best residues were substituted at contact positions 13, 14, and 15. In the other variant the best residues were substituted at all contact positions except the reactive site where a Gly was substituted. The four designed variants were: for PPE, T¹³E¹⁴Y¹⁵-OMTKY3 and T¹³E¹⁴Y¹⁵G¹⁸M²¹P³²V³⁶-OMTKY3, and for SGPB, S¹³D¹⁴Y¹⁵-OMTKY3 and S¹³D¹⁴Y¹⁵G¹⁸I¹⁹K²¹-OMTKY3. The free energies of association (ΔG⁰) of expressed variants have been measured with the proteases for which they were designed as well as with five other serine proteases and the results are discussed.     

Comparative genomic analysis of Helicobacter pylori from Malaysia identifies three distinct lineages suggestive of differential evolution

The discordant prevalence of Helicobacter pylori and its related diseases, for a long time, fostered certain enigmatic situations observed in the countries of the southern world. Variation in H. pylori infection rates and disease outcomes among different populations in multi-ethnic Malaysia provides a unique opportunity to understand dynamics of host–pathogen interaction and genome evolution. In this study, we extensively analyzed and compared genomes of 27 Malaysian H. pylori isolates and identified three major phylogeographic lineages: hspEastAsia, hpEurope and hpSouthIndia. The analysis of the virulence genes within the core genome, however, revealed a comparable pathogenic potential of the strains. In addition, we identified four genes limited to strains of East-Asian lineage. Our analyses identified a few strain-specific genes encoding restriction modification systems and outlined 311 core genes possibly under differential evolutionary constraints, among the strains representing different ethnic groups. The cagA and vacA genes also showed variations in accordance with the host genetic background of the strains. Moreover, restriction modification genes were found to be significantly enriched in East-Asian strains. An understanding of these variations in the genome content would provide significant insights into various adaptive and host modulation strategies harnessed by H. pylori to effectively persist in a host-specific manner.     

Stem cell therapy: A paradigm shift in breast cancer treatment

As per the latest Globocan statistics, the high prevalence rate of breast cancer in low- and middle-income countries has led to it becoming the most common cancer to be diagnosed, hence posing a major public health challenge. As per this data, more than 11.7% of the estimated new cancer cases in 2020 were due to breast cancer. A small but significant subpopulation of cells with self- renewing ability are present in the tumor stroma and have been given the nomenclature of cancer stem cells (CSCs). These cells display a high degree of plasticity owing to their ability to transition from the slowly cycling quiescent phase to the actively proliferating phenotype. This attribute of CSCs allows them to differentiate into various cell types having diverse functions. Breast CSCs have a pivotal role in development, metastasis, treatment resistance and relapse of breast cancers. This review focuses on the pathways regulating breast CSC maintenance and the current strategies that are being explored for directing the development of novel, targeted, therapeutic approaches for limiting and eradicating this aberrant stem cell population.     

The IkappaB kinase (IKK) inhibitor, NEMO-binding domain peptide, blocks osteoclastogenesis and bone erosion in inflammatory arthritis

Activation of NF-kappaB leads to expression of ample genes that regulate inflammatory and osteoclastogenic responses. The process is facilitated by induction of IkappaB kinase (IKK) complex that phosphorylates IkappaB and leads to its dissociation from the NF-kappaB complex, thus permitting activation of NF-kappaB. The IKK complex contains primarily IKKalpha, IKKbeta, and the regulatory kinase IKKgamma, also known as NEMO. NEMO regulates the IKK complex activity through its binding to carboxyl-terminal region of IKKalpha and IKKbeta, termed NEMO-binding domain (NBD). In this regard, a cell-permeable NBD peptide has been shown to block association of NEMO with the IKK complex and inhibit activation of NF-kappaB. Given the pivotal role of cytokine-induced NF-kappaB in osteoclastogenesis and inflammatory bone loss, we deduced that cell-permeable TAT-NBD peptide may hinder osteoclastogenesis and bone erosion in inflammatory arthritis. Using NBD peptides, we show that wild type, but not mutant, NBD blocks IKK activation and reduces cytokine-induced promoter and DNA binding activities of NF-kappaB and inhibits cytokine-induced osteoclast formation by osteoclast precursors. Consistent with the key role of NF-kappaB in osteoinflammatory responses in vivo, wild type TAT-NBD administered into mice prior to induction of inflammatory arthritis efficiently block in vivo osteoclastogenesis, inhibits focal bone erosion, and ameliorates inflammatory responses in the joints of arthritic mice. The mutant NBD peptide fails to exert these functions. These results provide strong evidence that IKKs are potent regulators of cytokine-induced osteoclastogenesis and inflammatory arthritis. More importantly, blockade of NEMO assembly with the IKK complex is a viable strategy to avert inflammatory osteolysis.     

Bimodular Peptide Synthetase SidE Produces Fumarylalanine in the Human Pathogen Aspergillus fumigatus

The filamentous mold Aspergillus fumigatus causes invasive aspergillosis, a potentially life-threatening infectious disease, in humans. The sidE gene encodes a bimodular peptide synthetase and was shown previously to be strongly upregulated during initiation of murine lung infection. In this study, we characterized the two adenylation domains of SidE with the ATP-[³²P]pyrophosphate exchange assay in vitro, which identified fumarate and l-alanine, respectively, as the preferred substrates. Using full-length holo-SidE, fumarylalanine (FA) formation was observed in vitro. Furthermore, FA was identified in A. fumigatus culture supernatants under inducing conditions, unless sidE was genetically inactivated. As FA is structurally related to established pharmaceutical products exerting immunomodulatory activity, this work may contribute to our understanding of the virulence of A. fumigatus.     

Mechanism of inhibition of PP2A activity and abnormal hyperphosphorylation of tau by I2(PP2A)/SET

Protein phosphatase-2A (PP2A) activity, which is compromised in Alzheimer disease brain, is regulated by two endogenous inhibitors, one of them being I(2)(PP2A), a 277 amino acid long protein also known as SET. Here we report that both the amino terminal fragment (I(2NTF); aa 1-175) and the carboxy terminal fragment (I(2CTF); aa 176-277) of I(2)(PP2A) inhibit PP2A by binding to its catalytic subunit PP2Ac and cause hyperphosphorylation of tau. The C-terminal acidic region in I(2CTF) and Val 92 in I(2NTF) are essential for their association with PP2Ac and inhibition of the phosphatase activity.     

Logged peat swamp forest supports greater macrofungal biodiversity than large‐scale oil palm plantations and smallholdings

Intensive land expansion of commercial oil palm agricultural lands results in reducing the size of peat swamp forests, particularly in Southeast Asia. The effect of this land conversion on macrofungal biodiversity is, however, understudied. We quantified macrofungal biodiversity by identifying mushroom sporocarps throughout four different habitats; logged peat swamp forest, large‐scale oil palm plantation, monoculture, and polyculture smallholdings. We recorded a total of 757 clusters of macrofungi belonging to 127 morphospecies and found that substrates for growing macrofungi were abundant in peat swamp forest; hence, morphospecies richness and macrofungal clusters were significantly greater in logged peat swamp forest than converted oil palm agriculture lands. Environmental factors that influence macrofungi in logged peat swamp forests such as air temperature, humidity, wind speed, soil pH, and soil moisture were different from those in oil palm plantations and smallholdings. We conclude that peat swamp forests are irreplaceable with respect to macrofungal biodiversity. They host much greater macrofungal biodiversity than any of the oil palm agricultural lands. It is imperative that further expansion of oil palm plantation into remaining peat swamp forests should be prohibited in palm oil producing countries. These results imply that macrofungal distribution reflects changes in microclimate between habitats and reduced macrofungal biodiversity may adversely affect decomposition in human‐modified landscapes.     

Distinct roles for intra- and extracellular siderophores during Aspergillus fumigatus infection

Siderophore biosynthesis by the highly lethal mould Aspergillus fumigatus is essential for virulence, but non-existent in humans, presenting a rare opportunity to strategize therapeutically against this pathogen. We have previously demonstrated that A. fumigatus excretes fusarinine C and triacetylfusarinine C to capture extracellular iron, and uses ferricrocin for hyphal iron storage. Here, we delineate pathways of intra- and extracellular siderophore biosynthesis and show that A. fumigatus synthesizes a developmentally regulated fourth siderophore, termed hydroxyferricrocin, employed for conidial iron storage. By inactivation of the nonribosomal peptide synthetase SidC, we demonstrate that the intracellular siderophores are required for germ tube formation, asexual sporulation, resistance to oxidative stress, catalase A activity, and virulence. Restoration of the conidial hydroxyferricrocin content partially rescues the virulence of the apathogenic siderophore null mutant Delta sidA, demonstrating an important role for the conidial siderophore during initiation of infection. Abrogation of extracellular siderophore biosynthesis following inactivation of the acyl transferase SidF or the nonribosomal peptide synthetase SidD leads to complete dependence upon reductive iron assimilation for growth under iron-limiting conditions, partial sensitivity to oxidative stress, and significantly reduced virulence, despite normal germ tube formation. Our findings reveal distinct cellular and disease-related roles for intra- and extracellular siderophores during mammalian Aspergillus infection.