Freezing/thawing effect on sewage sludge degradation and electricity generation in microbial fuel cell.

The effect of sludge freezing/thawing on its disintegration and subsequent use as substrate in a microbial fuel cell (MFC) was investigated to enhance organic matter degradation and electricity generation. Experimental results indicated that long freezing time (more than 48 h) was effective in disintegrating the sludge collected from the secondary sedimentation tank of a wastewater treatment plant. Freezing/thawing pretreatment could enhance the degradation of total chemical oxygen demand (COD) and electricity generation in MFC due to the higher concentration of soluble COD and ammonium nitrogen available in the pretreated sludge. The removal efficiency of total COD was increased from 25.3% (raw sludge as substrate) to 66.2% and the maximum power output was increased from 8.9 (raw sludge as substrate) to 10.2 W/m³ in MFC.

Fusarium sacchari hypovirus 1, a Member of Hypoviridae with Virulence Attenuation Capacity in Phytopathogenic Fusarium Species.

In a survey of mycoviruses in Fusarium species that cause sugarcane Pokkah boeng disease, twelve Fusarium strains from three Fusarium species (F. sacchari, F. andiyazi, and F. solani) were found to contain Fusarium sacchari hypovirus 1 (FsHV1), which we reported previously. The genomes of these variants range from 13,966 to 13,983 nucleotides, with 98.6% to 99.9% nucleotide sequence identity and 98.70% to 99.9% protein sequence similarity. Phylogenetic analysis placed these FsHV1 variants within the Alphahypovirus cluster of Hypoviridae. Intriguingly, no clear correlation was found between the geographic origin and host specificity of these viral variants. Additionally, six out of the twelve variants displayed segmental deletions of 1.5 to 1.8 kilobases, suggesting the existence of defective viral dsRNA. The presence of defective viral dsRNA led to a two-thirds reduction in the dsRNA of the wild-type viral genome, yet a tenfold increase in the total viral dsRNA content. To standardize virulence across natural strains, all FsHV1 strains were transferred into a single, virus-free Fusarium recipient strain, FZ06-VF, via mycelial fusion. Strains of Fusarium carrying FsHV1 exhibited suppressed pigment synthesis, diminished microspore production, and a marked decrease in virulence. Inoculation tests revealed varying capacities among different FsHV1 variants to modulate fungal virulence, with the strain harboring the FsHV1-FSA1 showing the lowest virulence, with a disease severity index (DSI) of 3.33, and the FsHV1-FS1 the highest (DSI = 17.66). The identification of highly virulent FsHV1 variants holds promise for the development of biocontrol agents for Pokkah boeng management.

Redundant and Distinct Roles of Two 14-3-3 Proteins in Fusarium sacchari, Pathogen of Sugarcane Pokkah Boeng Disease.

Fusarium sacchari, a key pathogen of sugarcane, is responsible for the Pokkah boeng disease (PBD) in China. The 14-3-3 proteins have been implicated in critical developmental processes, including dimorphic transition, signal transduction, and carbon metabolism in various phytopathogenic fungi. However, their roles are poorly understood in F. sacchari. This study focused on the characterization of two 14-3-3 protein-encoding genes, FsBmh1 and FsBmh2, within F. sacchari. Both genes were found to be expressed during the vegetative growth stage, yet FsBmh1 was repressed at the sporulation stage in vitro. To elucidate the functions of these genes, the deletion mutants ΔFsBmh1 and ΔFsBmh2 were generated. The ΔFsBmh2 exhibited more pronounced phenotypic defects, such as impaired hyphal branching, septation, conidiation, spore germination, and colony growth, compared to the ΔFsBmh1. Notably, both knockout mutants showed a reduction in virulence, with transcriptome analysis revealing changes associated with the observed phenotypes. To further investigate the functional interplay between FsBmh1 and FsBmh2, we constructed and analyzed mutants with combined deletion and silencing (ΔFsBmh/siFsBmh) as well as overexpression (O-FsBmh). The combinations of ΔFsBmh1/siFsBmh2 or ΔFsBmh2/siFsBmh1 displayed more severe phenotypes than those with single allele deletions, suggesting a functional redundancy between the two 14-3-3 proteins. Yeast two-hybrid (Y2H) assays identified 20 proteins with pivotal roles in primary metabolism or diverse biological functions, 12 of which interacted with both FsBmh1 and FsBmh2. Three proteins were specifically associated with FsBmh1, while five interacted exclusively with FsBmh2. In summary, this research provides novel insights into the roles of FsBmh1 and FsBmh2 in F. sacchari and highlights potential targets for PBD management through the modulation of FsBmh functions.

Structural basis of negative regulation of CRISPR-Cas7-11 by TPR-CHAT.

CRISPR‒Cas7-11 is a Type III-E CRISPR-associated nuclease that functions as a potent RNA editing tool. Tetratrico-peptide repeat fused with Cas/HEF1-associated signal transducer (TPR-CHAT) acts as a regulatory protein that interacts with CRISPR RNA (crRNA)-bound Cas7-11 to form a CRISPR-guided caspase complex (Craspase). However, the precise modulation of Cas7-11's nuclease activity by TPR-CHAT to enhance its utility requires further study. Here, we report cryo-electron microscopy (cryo-EM) structures of Desulfonema ishimotonii (Di) Cas7-11-crRNA, complexed with or without the full length or the N-terminus of TPR-CHAT. These structures unveil the molecular features of the Craspase complex. Structural analysis, combined with in vitro nuclease assay and electrophoretic mobility shift assay, reveals that DiTPR-CHAT negatively regulates the activity of DiCas7-11 by preventing target RNA from binding through the N-terminal 65 amino acids of DiTPR-CHAT (DiTPR-CHATNTD). Our work demonstrates that DiTPR-CHATNTD can function as a small unit of DiCas7-11 regulator, potentially enabling safe applications to prevent overcutting and off-target effects of the CRISPR‒Cas7-11 system.

Real-time sensing of neurotransmitters by functionalized nanopores embedded in a single live cell.

Interface between neuron cells and biomaterials is the key to real-time sensing, transmitting and manipulating of neuron activities, which are the long-term pursue of scientists and gain intense research focus recently. It is of great interest to develop a sensor with exquisite sensitivity and excellent selectivity for real-time monitoring neurotransmitters transport through single live cell. Sensing techniques including electrode-based methods, optogenetics, and nanowire cell penetration systems have been developed to monitor the neuron activities. However, their biocompatibilities remain a challenge. Protein nanopores with membrane compatibility and lumen tunability provide real-time, single-molecule sensitivities for biosensing of DNA, RNA, peptides and small molecules. In this study, an engineered protein nanopore MspA (Mycobacterium smegmatis porin A) through site-directed mutation with histidine selectively bind with Cu2+ in its internal lumen. Chelation of neurotransmitters such as L-glutamate (L-Glu), dopamine (DA) and norepinephrine (NE) with the Cu2+ creates specific current signals, showing different transient current blockade and dwell time in single channel electrophysiological recording. Furthermore, the functionalized M2MspA-N91H nanopores have been embedded in live HEK293T cell membrane for real-time, in situ monitoring of extracellular L-glutamate translocating through the nanopore. This biomimetic neurotransmitter nanopore has provided a new platform for future development of neuron sensors, drug carrier and artificial synapse.

Structural insights into outer membrane asymmetry maintenance in Gram-negative bacteria by MlaFEDB.

The highly asymmetric outer membrane of Gram-negative bacteria functions in the defense against cytotoxic substances, such as antibiotics. The Mla pathway maintains outer membrane lipid asymmetry by transporting phospholipids between the inner and outer membranes. It comprises six Mla proteins, MlaFEDBCA, including the ABC transporter MlaFEDB, which functions via an unknown mechanism. Here we determine cryo-EM structures of Escherichia coli MlaFEDB in an apo state and bound to phospholipid, ADP or AMP-PNP to a resolution of 3.3-4.1 Å and establish a proteoliposome-based transport system that includes MlaFEDB, MlaC and MlaA-OmpF to monitor the transport direction of phospholipids. In vitro transport assays and in vivo membrane permeability assays combined with mutagenesis identify functional residues that not only recognize and transport phospholipids but also regulate the activity and structural stability of the MlaFEDB complex. Our results provide mechanistic insights into the Mla pathway, which could aid antimicrobial drug development.

Layer-Dependent Properties of Ultrathin GeS Nanosheets and Application in UV-Vis Photodetectors.

Two-dimensional germanium sulfide (GeS), an analogue of phosphorene, has attracted broad attention owing to its excellent environmental stabilities, fascinating electronic and optical properties, and applications in various nanodevices. In spite of the current achievements on 2D GeS, the report of ultrathin few-layer GeS nanosheets within 5 nm is still lacking. Here in this contribution, we have achieved preparation of ultrathin few-layer GeS nanosheets with thicknesses of 1.3 ± 0.1 nm [approximately three layers (∼3L)], 3.2 ± 0.2 nm (∼6L), and 4.2 ± 0.3 nm (∼8L) via a typical liquid-phase exfoliation (LPE) method. Based on various experimental characterizations and first-principles calculations, the layer-dependent electronic, transport, and optical properties are investigated. For the few-layer GeS nanosheets, enhanced light absorption in the UV-vis region and superior photoresponse behavior with increasing layer number is observed, while for the thin films above 10 nm, the properties degenerate to the bulk feature. In addition, the as-prepared ultrathin nanosheets manifest great potential in the applications of photoelectrochemical (PEC)-type photodetectors, exhibiting excellent and stable periodic photoresponse behavior under the radiation of white light. The ∼8L GeS-based photodetector exhibits superior performance than the thinner GeS nanosheets (<4 nm), even better as compared to the bulk or film (above 10 nm) counterparts in terms of higher photoresponsivity along with remarkable photodetection performance in the UV-vis region. This work not only provides direct and solid evidence of the layer-number evolutionary band structure, mobility, and optical properties of ultrathin 2D GeS nanosheets but also promotes the foreseeable applications of 2D GeS as energy-related photoelectric devices.

Active DNA unwinding and transport by a membrane-adapted helicase nanopore.

Nanoscale transport through nanopores and live-cell membranes plays a vital role in both key biological processes as well as biosensing and DNA sequencing. Active translocation of DNA through these nanopores usually needs enzyme assistance. Here we present a nanopore derived from truncated helicase E1 of bovine papillomavirus (BPV) with a lumen diameter of c.a. 1.3 nm. Cryogenic electron microscopy (cryo-EM) imaging and single channel recording confirm its insertion into planar lipid bilayer (BLM). The helicase nanopore in BLM allows the passive single-stranded DNA (ssDNA) transport and retains the helicase activity in vitro. Furthermore, we incorporate this helicase nanopore into the live cell membrane of HEK293T cells, and monitor the ssDNA delivery into the cell real-time at single molecule level. This type of nanopore is expected to provide an interesting tool to study the biophysics of biomotors in vitro, with potential applications in biosensing, drug delivery and real-time single cell analysis.