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Leber congenital amaurosis (LCA) is the most common cause of inherited retinal degeneration in children. LCA patients with RPE65 mutations show accelerated cone photoreceptor dysfunction and death, resulting in early visual impairment. It is therefore crucial to develop a robust therapy that not only compensates for lost RPE65 function but also protects photoreceptors from further degeneration. Here, we show that in vivo correction of an Rpe65 mutation by adenine base editor (ABE) prolongs the survival of cones in an LCA mouse model. In vitro screening of ABEs and sgRNAs enables the identification of a variant that enhances in vivo correction efficiency. Subretinal delivery of ABE and sgRNA corrects up to 40% of Rpe65 transcripts, restores cone-mediated visual function, and preserves cones in LCA mice. Single-cell RNA-seq reveals upregulation of genes associated with cone phototransduction and survival. Our findings demonstrate base editing as a potential gene therapy that confers long-lasting retinal protection.

Yalan Yang,1,* Dao Xin,2,* Huike Wang,1,* Lulu Guan,1 Xiangrui Meng,1 Taiying Lu,1 Xiwen Bai,3 Feng Wang1 1Department of Oncology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, People’s Republic of China; 2Department of Medical Oncology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, People’s Republic of China; 3Department of Translational Medicine, Nanchang University Queen Mary School, Nanchang, People’s Republic of China*These authors contributed equally to this workCorrespondence: Feng Wang, Department of Oncology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, People’s Republic of China, Email zzuwangfeng@zzu.edu.cnPurpose: Neoadjuvant immunochemotherapy (nICT) for resectable locally advanced esophageal squamous cell carcinoma (LA-ESCC) has attracted widespread attention recently, whose safety and clinical benefit was observed in clinical researches. This study aimed to develop and validate a novel predictor systemic inflammation-tumor markers index (SITI) to predict the pathological complete response (pCR) for resectable LA-ESCC patients receiving nICT.Patients and Methods: A total of 147 LA-ESCC patients who underwent nICT followed by surgery from February 2020 to April 2022 were included in the study. The dynamic change of inflammatory indexes was compared at baseline, after two cycles of nICT and postoperative one month. Least absolute shrinkage and selection operator (LASSO) regression was performed to avoid collinearity and identify key indexes, with SITI constructed. After univariate and multivariate stepwise forward logistic analyses, a nomogram for pCR prediction was developed.Results: 41(27.9%) patients achieved pCR among 147 resectable LA-ESCC patients received nICT. Compared with baseline, most inflammatory indexes were significantly decreased at postoperative one month. 5 key indexes were identified and then a predictive index named SITI was constructed. The result showed that lower SITI and earlier clinical tumor node metastasis (cTNM) stage were more likely to achieve pCR. The nomogram for pCR prediction had excellent discrimination performance (C-index = 0.791).Conclusion: The SITI is an independent predictor for pCR in resectable LA-ESCC patients received nICT. To our knowledge, our nomogram is the first model using systemic inflammation-tumor markers for pCR prediction and may be a promising predictor to effectively differentiate pCR for nICT in LA-ESCC patients.Keywords: esophageal squamous cell carcinoma, neoadjuvant therapy, immunotherapy, pathologic complete response, inflammation, nomogram

Leber congenital amaurosis is caused by mutations in RPE65 and leads to retinal degeneration in children. Here, the authors show that in vivo base editing can prolong the survival of cone photoreceptors and rescue their function in a mouse model of the disease.

The epithelial-mesenchymal transition (EMT) is a fundamental developmental process that is abnormally activated in cancer metastasis. Dynamic changes in alternative splicing occur during EMT. ESRP1 and hnRNPM are splicing regulators that promote an epithelial splicing program and a mesenchymal splicing program, respectively. The functional relationships between these splicing factors in the genome scale remain elusive. Comparing alternative splicing targets of hnRNPM and ESRP1 revealed that they coregulate a set of cassette exon events, with the majority showing discordant splicing regulation. Discordant splicing events regulated by hnRNPM show a positive correlation with splicing during EMT; however, concordant events do not, indicating the role of hnRNPM in regulating alternative splicing during EMT is more complex than previously understood. Motif enrichment analysis near hnRNPM-ESRP1 coregulated exons identifies guanine-uridine rich motifs downstream from hnRNPM-repressed and ESRP1-enhanced exons, supporting a general model of competitive binding to these cis-elements to antagonize alternative splicing. The set of coregulated exons are enriched in genes associated with cell migration and cytoskeletal reorganization, which are pathways associated with EMT. Splicing levels of coregulated exons are associated with breast cancer patient survival and correlate with gene sets involved in EMT and breast cancer subtyping. This study identifies complex modes of interaction between hnRNPM and ESRP1 in regulation of splicing in disease-relevant contexts.

Hongfei Ke1 ,* Dexiang Liu2 ,* Tingting Li,1 Xili Chu,1 Danqing Xin,1 Min Han,1 Shuanglian Wang,1 Zhen Wang1 1Department of Physiology, School of Basic Medical Sciences, Cheeloo College of Medicine, Shandong University, Jinan, Shandong 250012, People’s Republic of China; 2Department of Medical Psychology and Ethics, School of Basic Medicine Sciences, Cheeloo College of Medicine, Shandong University, Jinan 250012, Shandong, People’s Republic of China*These authors contributed equally to this workCorrespondence: Zhen WangDepartment of Physiology, School of Basic Medical Sciences, Cheeloo College of Medicine, Shandong University, Jinan, Shandong 250012, People’s Republic of ChinaEmail wangzhen@sdu.edu.cnIntroduction: We have reported previously that hydrogen-rich saline (HS) plays a neuroprotective role in hypoxia-ischemia (HI) brain damage in newborn mice. However, the mechanisms for this neuroprotection resulting from HS remain unknown. In this study, we examined the potential for HS to exert effects upon microglial phagocytosis via involvement of the Akt signaling pathway as one of the neuroprotective mechanisms in response to neonatal HI.Methods: The HI brain injury model was performed on postnatal day (PND) 7 (modified Vannucci model). The acute brain damage was detected at 3 days after HI exposure. The behavioral and functional screening of the pups at PND11 and PND13 and their long-term outcomes (PND35, 28-days post-HI) were evaluated sensorimotor performance and cognitive functions, respectively.Results: The result showed that HS administration alleviated HI-induced edema, infract volume and cellular apoptosis within the cortex of neonatal mice. Accompanying these indices of neuroprotection from HS were reductions in HI-induced phagocytosis in microglia as demonstrated in vivo and in vitro, effects that were associated with increasing levels of Akt phosphorylation and improvements in neurobehavioral responses. These beneficial effects of HS were abolished in mice treated with an Akt inhibitor.Discussion: These results demonstrate that HS treatment attenuates neurobehavioral deficits and apoptosis resulting from HI, effects which were associated with reductions in phagocytosis and appear to involve the Akt signaling pathway.Keywords: microglia, phagocytosis, hypoxia-ischemia, HS, Akt

Min Han,1– 3 Ying Cao,2 Hao Xue,1 Xili Chu,2 Tingting Li,2 Danqing Xin,2 Lin Yuan,2 Hongfei Ke,2 Gang Li,1 Zhen Wang2 1Department of Neurosurgery, Qilu Hospital, Cheeloo College of Medicine, Shandong University, Jinan, Shandong 250012, People’s Republic of China; 2Department of Physiology, School of Basic Medical Sciences, Cheeloo College of Medicine, Shandong University, Jinan, Shandong 250012, People’s Republic of China; 3Department of Neurosurgery, The Fifth People’s Hospital of Jinan, Jinan, Shandong Province 250022, People’s Republic of ChinaCorrespondence: Zhen WangDepartment of Physiology, School of Basic Medical Sciences, Cheeloo College of Medicine, Shandong University, Jinan, Shandong 250012, People’s Republic of ChinaEmail wangzhen@sdu.edu.cnGang LiDepartment of Neurosurgery, Qilu Hospital, Cheeloo College of Medicine, Shandong University, Jinan, Shandong 250012, People’s Republic of ChinaEmail dr.ligang@sdu.edu.cnIntroduction: Cerebral ischemia-reperfusion injury (CIRI) is the main factor that leads to poor prognosis of cerebral ischemia. Apoptosis has been shown to occur during the process of CIRI. Extracellular vesicles derived from mesenchymal stromal cells (MSCs-EVs) have shown broad potential for treating brain dysfunction and eliciting neuroprotective effects after stroke through neurogenesis and angiogenesis. However, the mechanism of action of extracellular vesicles during CIRI is not well known.Methods: A middle cerebral artery occlusion (MCAO) model was induced by the modified Longa method, and MSCs-EVs were injected via the tail vein.Results: Our results showed that MSCs-EVs significantly alleviated neurological deficits, reduced the volume of cerebral infarction and brain water content, improved pathological lesions in cortical brain tissue, and attenuated neuronal apoptosis in the cortex at 24 h and 48 h after MCAO in rats. Western blotting analysis showed that MSCs-EVs significantly upregulated p-AMPK and downregulated p-JAK2, p-STAT3 and p-NF-κB. In addition, an AMPK pathway blocker reversed the effect of MSCs-EVs on brain damage.Conclusion: These results indicate that MSCs-EVs protected MCAO-injured rats, possibly by regulating the AMPK and JAK2/STAT3/NF-κB signaling pathways. This study supports the use of MSCs-EVs as a potential treatment strategy for MCAO in the future.Keywords: extracellular vesicles, cerebral ischemia reperfusion, neuroprotection, AMPK, nuclear factor κB, JAK2/STAT3 signaling pathway

Tumor metastasis remains the major cause of cancer-related death, but its molecular basis is still not well understood. Here we uncovered a splicing-mediated pathway that is essential for breast cancer metastasis. We show that the RNA-binding protein heterogeneous nuclear ribonucleoprotein M (hnRNPM) promotes breast cancer metastasis by activating the switch of alternative splicing that occurs during epithelial-mesenchymal transition (EMT). Genome-wide deep sequencing analysis suggests that hnRNPM potentiates TGFβ signaling and identifies CD44 as a key downstream target of hnRNPM. hnRNPM ablation prevents TGFβ-induced EMT and inhibits breast cancer metastasis in mice, whereas enforced expression of the specific CD44 standard (CD44s) splice isoform overrides the loss of hnRNPM and permits EMT and metastasis. Mechanistically, we demonstrate that the ubiquitously expressed hnRNPM acts in a mesenchymal-specific manner to precisely control CD44 splice isoform switching during EMT. This restricted cell-type activity of hnRNPM is achieved by competition with ESRP1, an epithelial splicing regulator that binds to the same cis-regulatory RNA elements as hnRNPM and is repressed during EMT. Importantly, hnRNPM is associated with aggressive breast cancer and correlates with increased CD44s in patient specimens. These findings demonstrate a novel molecular mechanism through which tumor metastasis is endowed by the hnRNPM-mediated splicing program.

Chuanliang Zhao,1–3 Xin Zhou,1,2 Jie Qiu,1,2 Danqing Xin,2 Tingting Li,2 Xili Chu,2 Hongtao Yuan,2 Haifeng Wang,1 Zhen Wang,2 Dachuan Wang1 1Department of Spinal Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong, People’s Republic of China; 2Department of Physiology, Shandong University School of Basic Medical Sciences, Jinan, Shandong, People’s Republic of China; 3Department of Orthopedic, Feicheng Hospital of Traditional Chinese Medicine, Feicheng, Shandong, People’s Republic of ChinaCorrespondence: Dachuan WangDepartment of Spinal Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong, People’s Republic of ChinaTel +86 139 5316 9759Email wangdachuan@medmail.com.cnPurpose: Spinal cord injury (SCI) is a relatively common, devastating traumatic condition resulting in permanent disability. In this study, the use of exosomes derived from bone mesenchymal stem cells (BMSCs-Exo) as a cell-free therapy for the treatment of SCI in rats was investigated to gain insights into their mechanisms of action.Methods: Rats were randomly divided into three groups, Sham (treated with PBS), SCI (SCI injury + PBS) and SCI + Exo (SCI injury + BMSCs-Exo). Changes in the complement system between the three groups were assessed with the use of proteomics. The proteomic data were verified using reverse transcription-polymerase chain reaction (RT-PCR). In addition, the distributions of BMSCs-Exo in rats with SCI were detected by immunofluorescence. Moreover, SCI-activated NF-κB levels were determined using Western blot.Results: SCI insult increased complement levels, including C4, C5, C6, C4 binding protein alpha and complement factor H. In contrast, the SCI + BMSCs-Exo group exhibited attenuated SCI-induced complement levels. Immunofluorescence assay results revealed that BMSCs-Exo mainly accumulated at the spinal cord injury site and were bound to microglia cells. Western blot analysis of tissue lysates showed that BMSCs-Exo treatment also inhibited SCI-activated nuclear factor kappa-B (NF-κB).Conclusion: BMSCs-Exo play a protective role in spinal cord injury by inhibiting complement mRNA synthesis and release and by inhibiting SCI-activated NF-κB by binding to microglia.Keywords: exosomes, proteomics, complement, spinal cord injury

Abstract Background The development of CRISPR genome editing has transformed biomedical research. Most applications reported thus far rely upon the Cas9 protein from Streptococcus pyogenes SF370 (SpyCas9). With many RNA guides, wildtype SpyCas9 can induce significant levels of unintended mutations at near-cognate sites, necessitating substantial efforts toward the development of strategies to minimize off-target activity. Although the genome-editing potential of thousands of other Cas9 orthologs remains largely untapped, it is not known how many will require similarly extensive engineering to achieve single-site accuracy within large genomes. In addition to its off-targeting propensity, SpyCas9 is encoded by a relatively large open reading frame, limiting its utility in applications that require size-restricted delivery strategies such as adeno-associated virus vectors. In contrast, some genome-editing-validated Cas9 orthologs are considerably smaller and therefore better suited for viral delivery. Results Here we show that wildtype NmeCas9, when programmed with guide sequences of the natural length of 24 nucleotides, exhibits a nearly complete absence of unintended editing in human cells, even when targeting sites that are prone to off-target activity with wildtype SpyCas9. We also validate at least six variant protospacer adjacent motifs (PAMs), in addition to the preferred consensus PAM (5′-N4GATT-3′), for NmeCas9 genome editing in human cells. Conclusions Our results show that NmeCas9 is a naturally high-fidelity genome-editing enzyme and suggest that additional Cas9 orthologs may prove to exhibit similarly high accuracy, even without extensive engineering.

ABSTRACT In their natural settings, CRISPR-Cas systems play crucial roles in bacterial and archaeal adaptive immunity to protect against phages and other mobile genetic elements, and they are also widely used as genome engineering technologies. Previously we discovered bacteriophage-encoded Cas9-specific anti-CRISPR (Acr) proteins that serve as countermeasures against host bacterial immunity by inactivating their CRISPR-Cas systems (A. Pawluk, N. Amrani, Y. Zhang, B. Garcia, et al., Cell 167:1829–1838.e9, 2016, https://doi.org/10.1016/j.cell.2016.11.017). We hypothesized that the evolutionary advantages conferred by anti-CRISPRs would drive the widespread occurrence of these proteins in nature (K. L. Maxwell, Mol Cell 68:8–14, 2017, https://doi.org/10.1016/j.molcel.2017.09.002; A. Pawluk, A. R. Davidson, and K. L. Maxwell, Nat Rev Microbiol 16:12–17, 2018, https://doi.org/10.1038/nrmicro.2017.120; E. J. Sontheimer and A. R. Davidson, Curr Opin Microbiol 37:120–127, 2017, https://doi.org/10.1016/j.mib.2017.06.003). We have identified new anti-CRISPRs using the same bioinformatic approach that successfully identified previous Acr proteins (A. Pawluk, N. Amrani, Y. Zhang, B. Garcia, et al., Cell 167:1829–1838.e9, 2016, https://doi.org/10.1016/j.cell.2016.11.017) against Neisseria meningitidis Cas9 (NmeCas9). In this work, we report two novel anti-CRISPR families in strains of Haemophilus parainfluenzae and Simonsiella muelleri, both of which harbor type II-C CRISPR-Cas systems (A. Mir, A. Edraki, J. Lee, and E. J. Sontheimer, ACS Chem Biol 13:357–365, 2018, https://doi.org/10.1021/acschembio.7b00855). We characterize the type II-C Cas9 orthologs from H. parainfluenzae and S. muelleri, show that the newly identified Acrs are able to inhibit these systems, and define important features of their inhibitory mechanisms. The S. muelleri Acr is the most potent NmeCas9 inhibitor identified to date. Although inhibition of NmeCas9 by anti-CRISPRs from H. parainfluenzae and S. muelleri reveals cross-species inhibitory activity, more distantly related type II-C Cas9s are not inhibited by these proteins. The specificities of anti-CRISPRs and divergent Cas9s appear to reflect coevolution of their strategies to combat or evade each other. Finally, we validate these new anti-CRISPR proteins as potent off-switches for Cas9 genome engineering applications. IMPORTANCE As one of their countermeasures against CRISPR-Cas immunity, bacteriophages have evolved natural inhibitors known as anti-CRISPR (Acr) proteins. Despite the existence of such examples for type II CRISPR-Cas systems, we currently know relatively little about the breadth of Cas9 inhibitors, and most of their direct Cas9 targets are uncharacterized. In this work we identify two new type II-C anti-CRISPRs and their cognate Cas9 orthologs, validate their functionality in vitro and in bacteria, define their inhibitory spectrum against a panel of Cas9 orthologs, demonstrate that they act before Cas9 DNA binding, and document their utility as off-switches for Cas9-based tools in mammalian applications. The discovery of diverse anti-CRISPRs, the mechanistic analysis of their cognate Cas9s, and the definition of Acr inhibitory mechanisms afford deeper insight into the interplay between Cas9 orthologs and their inhibitors and provide greater scope for exploiting Acrs for CRISPR-based genome engineering.

Background: Based on computed tomography images of the thoracolumbar intervertebral foramen and its surrounding parameters, and analysing the intervertebral foramen morphology and the correlation between the measured parameters, to provide an anatomical basis for clinical minimally invasive transvertebral surgery.Materials and Methods: Ten fresh adult cadaveric specimens (32-50 years old) with bilateral (T1-S1) spinal segments were selected for a total of 20 sides, a total of 340 intervertebral foramens and were measured with vernier callipers in the Department of Anatomy, Inner Mongolia Medical University. The intervertebral foramen height, the minimum sagittal diameter of the foramen, the width of the spinal ganglion, the sagittal diameter of the spinal ganglion and the sagittal diameter of the intervertebral foramen were measured. This study was reviewed and approved by the local Ethics Committee.Results: The results of the minimum sagittal diameter of the T9-10~L5/S1 intervertebral foramen were (6.93 ± 1.99) mm, (7.33 ± 1.44) mm, (7.41 ± 0.63) mm, (6.85 ± 1.08) mm, (6.79 ± 1.86) mm, (7.82 ± 3.25) mm, (8.23 ± 2.27) mm, (9.17 ± 2.33) mm, (8.38 ± 1.63) mm; the average height of the T2/3 to L5/S1 intervertebral space was (4.82 ± 1.88) mm, (3.95 ± 0.80) mm, (4.04 ± 0.52) mm, (4.26 ± 0.78) mm, (4.39 ± 1.16) mm, (5.15 ± 1.59) mm, (5.51 ± 1.49) mm, (5.97 ± 2.60) mm, (7.13 ± 2.07) mm, (8.94 ± 1.37) mm, (9.01 ± 1.47) mm, (11.63 ± 1.63) mm, (14.20 ± 1.37) mm, (14.22 ± 2.33) mm, (14.22 ± 2.33) mm, (13.32 ± 1.37) mm intervertebral foramen height, intervertebral foramen minimum sagittal diameter, spinal ganglion width, spinal ganglion sagittal diameter. P > 0.05 for comparison of the left and right sides of the intervertebral space, with no statistically significant difference. L4/5, L5/S1 segment left and right bilateral contrast with the middle height of the vertebral space p < 0.05, the difference is statistically significant. The remaining segments left and right bilaterally contrasted p > 0.05, and the difference was not statistically significant.Conclusions: The minimum height of intervertebral foramen in the thoracolumbar segment was T6/7, and L1/2 was the minimum height in the lumbar segment. When placing a spinal endoscopic working channel safely into intervertebral foramen, it is necessary to perform an enlarging foraminoplasty to reduce the risk of injury to the exiting nerve root. [ABSTRACT FROM AUTHOR]