Characterization of long-term ex vivo expansion of tree shrew spermatogonial stem cells

Tree shrews(Tupaia belangeri chinensis)share a close relationship to primates and have been widely used in biomedical research.We previously established a spermatogonial stem cell(SSC)-based gene editing platform to generate transgenic tree shrews.However,the influences of long-term expansion on tree shrew SSC spermatogenesis potential remain unclear.Here,we examined the in vivo spermatogenesis potential of tree shrew SSCs cultured across different passages.We found that SSCs lost spermatogenesis ability after long-term expansion(>50 passages),as indicated by the failure to colonize the seminiferous epithelium and generate donor spermatogonia(SPG)-derivedspermatocytesor spermatids marking spermatogenesis.RNA sequencing(RNA-seq)analysis of undifferentiated SPGs across different passages revealed significant gene expression changes after sub-culturing primary SPG lines for more than 40 passages on feeder layers.Specifically,DNA damage response and repair genes(e.g.,MRE11,SMC3,BLM,and GEN1)were down-regulated,whereas genes associated with mitochondrial function(e.g.,NDUFA9,NDUFA8,NDUFA13,and NDUFB8)were up-regulated after expansion.The DNA damage accumulation and mitochondrial dysfunction were experimentally validated in high-passage cells.Supplementation with nicotinamide adenine dinucleotide(NAD+)precursor nicotinamide riboside(NR)exhibited beneficial effects by reducing DNA damage accumulation and mitochondrial dysfunction in SPG elicited by long-term culture.Our research presents a comprehensive analysis of the genetic and physiological attributes critical for the sustained expansion of undifferentiated SSCs in tree shrews and proposes an effective strategy for extended in vitro maintenance.

Effect of photoperiod on development and reproduction of Megacopta cribraria(Hemiptera:Plataspidae)

Megacopta cribraria (Hemiptera: Plataspidae) continually outbreaks due to suitable photoperiod in recent years. Effect of photoperiod on growth, development and reproduction of M. cribraria were assessed in this study. Results indicated that developmental duration, nutrient accumulation efficiency, and adult fecundity of M. cribraria were significantly different under 6 photoperiodical conditions. Developmental duration of nymph stage gradually tended to be shorter as day time increase. Body weights of 5th instar nymphs for 16 h and 4 h day time photoperiods were 5.2 mg and 4.6 mg, respectively. Moreover, longevity of adults tended to be longer as day time increase. However, for the short day photoperiod (4 L:20 D and 8 L:16 D), population showed no reproductive behaviors. Index of population trend increased with photoperiod extension and adults showed stronger reproductive capacity and longer longevity. This research identified the favorable photoperiodical conditions before outbreak. It may provided reference for ecological adaptability of M. cribraria, and contribute to the scientific basis for forecasting and controlling of M. cribraria.

A novel missense variant in ACAA1 contributes to early-onset Alzheimer’s disease,impairs lysosomal function,and facilitates amyloid-p pathology and cognitive decline

Alzheimer’s disease(AD)is characterized by progressive synaptic dysfunction,neuronal death,and brain atrophy,with amyloid-p(Ap)plaque deposits and hyperphosphorylated tau neurofibrillary tangle accumulation in the brain tissue,which all lead to loss of cognitive function.Pathogenic mutations in the well-known AD causal genes including APP,PSEN1,and PSEN2 impair a variety of pathways,including protein processing,axonal transport,and metabolic homeostasis.Here we identified a missense variant rs117916664(c.896T>C,p.Asn299Ser[p.N299S])of the acetyl-CoA acyltransferase 1(ACM1)gene in a Han Chinese AD family by whole-genome sequencing and validated its association with early-onset familial AD in an independent cohort.Further in vitro and in vivo evidence showed that ACAA1 p.N299S contributes to AD by disturbing its enzymatic activity,impairing lysosomal function,and aggravating the Ap pathology and neuronal loss,which finally caused cognitive impairment in a murine model.Our findings reveal a fundamental role of peroxisome-mediated lysosomal dysfunction in AD pathogenesis.

Direct evidence of CRISPR-Cas9-mediated mitochondrial genome editing

Pathogenic mitochondrial DNA(mtDNA)mutations can cause a variety of human diseases.The recent development of genome-editing technologies to manipulate mtDNA,such as mitochondria-targeted DNA nucleases and base editors,offer a promising way for curing mitochondrial diseases caused by mtDNA mutations.The CRISPR-Cas9 system is a widely used tool for genome editing;however,its application in mtDNA editing is still under debate.In this study,we developed a mito-Cas9 system by adding the mitochondria-targeted sequences and 30 untranslated region of nuclear-encoded mitochondrial genes upstream and downstream of the Cas9 gene,respectively.