Yanming Dong

Mitochondria play a pivotal role in spermatogenesis and male fertility, in which the selective autophagic degradation of mitochondria (mitophagy) constitutes a key process. In this study, we identify mitochondrial carrier homolog 2 (MTCH2) as a novel and critical regulator of germ cell mitophagy. Localized to the mitochondrial outer membrane (OMM), MTCH2 directly interacts with the core autophagy protein BECN1, thereby inhibiting autophagic flux and specifically suppressing mitophagy. Germ cell-specific heterozygous deletion of Mtch2 was sufficient to cause profound spermatogenic defects, including disorganized seminiferous tubules, vacuolization, mitochondrial sheath abnormalities, and significantly impaired sperm fertilization capacity. Transcriptomic profiling revealed that MTCH2 deficiency dysregulates pathways central to mitochondrial function and programmed cell death. Importantly, augmenting autophagy ameliorated mitochondrial dysfunction induced by MTCH2 deficiency, as characterized by impaired mitochondrial membrane potential, elevated mitochondrial reactive oxygen species (mtROS) production and cellular ROS levels. Collectively, our findings establish MTCH2 as an essential negative regulator of mitophagy that partners with BECN1 to maintain mitochondrial homeostasis, thereby ensuring normal spermatogenesis and male fertility.

Nanobodies have gained attention as potential therapeutic and diagnostic agents for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) due to their ability to bind and neutralize the virus. However, rapid, scalable, and robust production of nanobodies for SARS-CoV-2 remains a crucial challenge. In this study, we developed a visual and high-efficiency biomanufacturing method for nanobodies with Escherichia coli by fusing the super-folder green fluorescent protein (sfGFP) to the N-terminus or C-terminus of the nanobody. Several receptor-binding domain (RBD)-specific nanobodies of the SARS-CoV-2 spike protein (S) were secreted onto the surface of E. coli cells and even into the culture medium, including Fu2, ANTE, mNb6, MR3-MR3, and n3113.1. The nanobodies secreted by E. coli retained equal activity as prior research, regardless of whether sfGFP was removed. Since some of the nanobodies bound to different regions of the RBD, we combined two nanobodies to improve the affinity. Fu2-sfGFP-ANTE was constructed to be bispecific for the RBD, and the bispecific nanobody exhibited significantly higher affinity than Fu2 (35.0-fold), ANTE (7.3-fold), and the combination of the two nanobodies (3.3-fold). Notably, Fu2-sfGFP-ANTE can be normally secreted into the culture medium and outer membrane. The novel nanobody production system enhances the efficiency of nanobody expression and streamlines the downstream purification process, enabling large-scale, cost-effective nanobody production. In addition, E. coli cells secreting the nanobodies on their surface facilitates screening and characterization of antigen-binding clones.

Hand, foot, and mouth disease (HFMD), caused by enteroviruses, mostly including EV71, CVA6, CVA10, and CVA16, is an acute infectious disease commonly found in children. Due to no approved antiviral therapies and available vaccines, except for EV71, developing accurate diagnostic methods of HFMD is essential for controlling its spread and mitigating its impact on public health. Here, we create a MIRA-HEV-PAND multiple nucleic acid typing method that utilizes PfAgo to identify enterovirus type A pathogens (EV71, CVA6, CVA10, and CVA16) and universal type EVU. The MDC (minimum detection concentration) level of MIRA-HEV-PAND is within the range of 1.66 aM (1.0 copy/μL), which was matched to that of qPCR assays and even more sensitive up to 10%. Importantly, the MIRA-HEV-PAND method exhibits higher sensitivity and less time-consuming efficiency compared to the approach that combines PCR amplification instead of MIRA amplification. Meanwhile, though the quintuple and single-tube multiple MIRA-HEV-PAND detection system can be used for one viral target or multiple viral target detection, the single-tube detection system detects more efficiently and rapidly than the quintuple-tube multiple detection system. Moreover, the diagnostic results obtained by evaluating clinical samples using MIRA-HEV-PAND show a complete consistency of 100% with qPCR assays. The MIRA-HEV-PAND method can screen a wider range of target regions using low-cost guide DNA without being limited to PAM sequences, compared to the MARPLES based on the CRISPR-Cas12a. The utilization of this correlation can be beneficial for the application of molecular testing for clinical diagnoses and the study of human enteroviruses A infection and virus typing on an epidemiological scale.

As a mosquito-borne flavivirus, Zika virus (ZIKV) has been identified as a global health threat. The virus has been linked to severe congenital disabilities, including microcephaly and other congenital malformations, resulting in fatal intrauterine death. Therefore, developing sensitive and specific methods for the early detection and accurate diagnosis of the ZIKV is essential for controlling its spread and mitigating its impact on public health. Herein, we set up a novel nucleic acid detection system based on Pyrococcus furiosus Argonaute (PfAgo)-mediated nucleic acid detection, targeting the non-structural protein 5 (NS5) region of the ZIKV genome (abbreviated ZIKV-PAND). Without preamplification with the polymerase chain reaction (PCR), the minimum detection concentration (MDC) of ZIKV-PAND was about 10 nM. When introducing an amplification step, the MDC can be dramatically decreased to the aM level (8.3 aM), which is comparable to qRT-PCR assay (1.6 aM). In addition, the diagnostic findings from the analysis of simulated clinical samples or Zika virus samples using ZIKV-PAND show a complete agreement of 100% with qRT-PCR assays. This correlation can aid in the implementation of molecular testing for clinical diagnoses and the investigation of ZIKV infection on an epidemiological scale.

Parvovirus B19 (B19V) is pathogenic to humans and causes various human diseases. However, no antiviral agents or vaccines currently exist for the treatment or prevention of B19V infection. Therefore, developing sensitive and specific methods for B19V infection diagnosis is essential for accurate diagnoses. Previously, a Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-Cas12a (cpf1)-based electrochemical biosensor (E-CRISPR) with a picomole sensitivity for B19V detection was established. Herein, we set up a novel nucleic acid detection system based on Pyrococcus furiosus Argonaute (PfAgo)-mediated nucleic acid detection, targeting the nonstructural protein 1 (NS1) region of the B19V viral genome (abbreviated B19-NS1 PAND). Benefiting from independent protospacer adjacent motif (PAM) sequences, PfAgo can recognize their target with guide DNA (gDNA) that is easy to design and synthesize at a low cost. In contrast to E-CRISPR, without preamplification with Polymerase Chain Reaction (PCR), the Minimum Detectable Concentration (MDC) of three guide- or single guide-mediated B19-NS1 PAND was about 4 nM, approximately 6-fold more than E-CRISPR. However, when introducing an amplification step, the MDC can be dramatically decreased to the aM level (54 aM). In addition, the diagnostic results from clinical samples with B19-NS1 PAND revealed 100% consistency with PCR assays and subsequent Sanger sequencing tests, which may assist in molecular testing for clinical diagnosis and epidemiological investigations of B19V.