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The CRISPR-Cas9 system is increasingly being used as a gene editing therapeutic technique in complex diseases but concerns remain regarding the clinical risks of Cas9 immunogenicity. In this study, we detected antibodies against

The purpose was to analyze the risk factors for implant loss after simultaneous guided bone regeneration (GBR).Patients who underwent implant placement with simultaneous GBR between January 2011 and December 2018 were screened for this study. The cumulative survival rate (CSR) was calculated using the life table method. Log-rank test and Kaplan-Meier survival estimates were used to identify potential risk factors for implant loss. The association between the investigated variables and implant loss was determined using hazard ratios (HRs) obtained from a multivariate Cox regression analysis.A total of 3973 patients with 5404 implants were included in this study. The CSRs of the implants at 1, 5, and 10 years were 99.6%, 98.9%, and 98.7%, respectively. Male patient (HR = 2.94, 95% CI: 1.41-6.14), periodontitis (HR = 4.26, 95% CI: 2.05-9.86), tissue-level implants (HR = 3.02, 95% CI: 1.30-6.98), narrow implants (HR = 2.71, 95% CI: 1.12-6.57), and implant length ≤10 mm (HR = 2.91, 95% CI: 1.41-6.02) significantly increased the risk of implant loss (p 0.05). The risk of implant loss was significantly higher in the maxillary posterior region (HR = 2.26, 95% CI: 1.04-4.90) than in the maxillary anterior region (p 0.05). Compared to Straumann, Nobel (HR = 4.07, 95% CI: 1.75-9.44) and other implant systems (HR = 14.23, 95% CI: 4.32-46.85) showed a significantly higher risk of implant loss (p 0.05).Male patient, periodontitis, maxillary posterior region, Nobel implant system, other implant systems, tissue-level implants, narrow implants, and implant length ≤10 mm were considered risk factors for implant loss after simultaneous GBR.

PURPOSE: To determine the application value of next-generation sequencing (NGS)-based preimplantation genetic testing for aneuploidies (PGT-A). METHODS: We conducted a retrospective case–control study on a cohort of frozen-thawed embryo transfer (FET) cycles following preimplantation genetic testing for monogenic disorders (PGT-M) between 2014 and 2017. Cycles that produced live births or early miscarriages were divided into live birth group (n = 76) or miscarriage group (n = 19), respectively. The NGS-based aneuploidy screening was performed on the multiple displacement amplification (MDA) products of the embryonic trophectoderm biopsy samples that were cryopreserved following PGT-M. RESULTS: In the live birth group, 75% (57/76) embryos were euploid and 14.5% (11/76) were aneuploid. The remaining 10.5% (8/76) embryos were NGS-classified mosaic with the high- (≥ 50%) and low-level (

PURPOSE: This study aimed to evaluate the value of long-read sequencing for preimplantation haplotype linkage analysis. METHODS: The genetic material of the three β-thalassemia mutation carrier couples was sequenced using single-molecule real-time sequencing in the 7.7-kb region of the HBB gene and a 7.4-kb region that partially overlapped with it to detect the presence of 17 common HBB gene mutations in the Chinese population and the haplotypes formed by the continuous array of single-nucleotide polymorphisms linked to these mutations. By using the same method to analyze multiple displacement amplification products of embryos from three families and comparing the results with those of the parents, it could be revealed whether the embryos carry disease-causing mutations without the need for a proband. RESULTS: The HBB gene mutations of the three couples were accurately detected, and the haplotype linked to the pathogenic site was successfully obtained without the need for a proband. A total of 68.75% (22/32) of embryos from the three families successfully underwent haplotype linkage analysis, and the results were consistent with the results of NGS-based mutation site detection. CONCLUSION: This study supports long-read sequencing as a potential tool for preimplantation haplotype linkage analysis. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10815-022-02415-1.

Which of the two mainstream endometrial preparation regimens, assisted natural cycle (NC) or hormone replacement treatment cycle (HRT), help frozen-thawed embryo transfer (FET) cycles after preimplantation genetic testing (PGT) achieve better clinical outcomes?This retrospective analysis included 3400 vitrified-warmed single blastocyst transfer cycles after PGT from January 2011 to November 2020, and involved 2332 patients with regular menstrual cycles. The decision to proceed with an assisted NC (n = 827) or HRT (n = 2573) before FET was reached based on a combination of patient preference and physician guidance. Clinical pregnancy rate, live birth rate, early miscarriage rate and obstetric outcomes were compared.No significant difference was observed between the assisted NC and HRT groups in terms of clinical pregnancy rate (51.6% versus 50.7%, P = 0.634), live birth rate (44.0% versus 43.4%, P = 0.746) or early miscarriage rate (12.6% versus 12.0%, P = 0.707). Multivariate analysis indicated that the endometrial preparation protocol was not an independent factor for a clinical pregnancy or live birth. In the HRT group, the Caesarean section rate (64.7% versus 51.9%, P 0.001) and pregnancy complication rate (20.2% versus 13.8%, P = 0.003) were significantly higher. The two groups were not statistically different with respect to gestational age, early preterm birth rate, fetal weight or fetal birth defect rate.For patients undergoing a PGT-FET cycle involving a single blastocyst transfer, using assisted NC and HRT for the endometrial preparation could lead to comparable rates of clinical pregnancy and live birth. Additionally, NC is safer than HRT in terms of avoiding pregnancy complications and adverse obstetric outcomes.

Objective: To explore the mechanism by which resveratrol (RES) affects the proliferation, migration, invasion and apoptosis of oral squamous cell carcinoma (OSCC) cells through inhibiting the Wnt pathway. Methods: Ki-67, matrix metalloproteinase-9 (MMP-9) and B-cell lymphoma-2 (Bcl-2) protein expression levels were detected by immunohistochemical staining. Tca8113 cells were treated with different concentrations of RES (0, 50, 100 and 200 μmol/L) and divided into control, low-dose RES, medium-dose RES and high-dose RES groups. EdU staining,Transwell migration and invasion assays and Annexin V-FITC/PI double-staining were employed to determine cell proliferation, migration, invasion and apoptosis, respectively. E-cadherin and N-cadherin mRNA expression levels in cells were analyzed by RT-qPCR. Wnt1,β-catenin and glycogen synthase kinase-3β (GSK-3β) protein expression levels were detected by Western blotting. Results: Ki-67, MMP-9 and Bcl-2 were positively expressed in OSCC tissues, with higher rates than those in normal oral mucosal tissues. RES significantly inhibited the proliferation, migration and invasion ability of Tca8113 cells, down-regulated the expression levels of N-cadherin mRNA and Wntl, β-catenin and GSK-3β proteins, promoted apoptosis, and up-regulated E-cadherin mRNA expression level dose-dependently (P Conclusion: RES can inhibit the proliferation, migration and invasion of OSCC cells and promote apoptosis possibly by inhibiting the Wnt signaling pathway, providing a potential target for targeted therapy of OSCC.

To analyze the marginal bone loss (ΔMBL) of tissue- or bone-level implants after placed with simultaneous guided bone regeneration (GBR).A total of 151 patients who received 104 tissue-level or 128 bone-level implants placement with simultaneous GBR in the mandibular posterior region between January 2011 and December 2016 were included in this study. The marginal bone level (MBL) was recorded using the radiographic data obtained at implant placement, second-stage surgery, and the follow-up visit. Generalized estimating equation (GEE) was used to compare the ΔMBL of tissue- and bone-level implants, and the influencing factors of ΔMBL were further analyzed.At the last follow-up visit, the MBL of tissue-level implants was 0.73 ± 0.86 mm, above the rough-smooth interface, while that of bone-level implants was 0.82 ± 1.05 mm, above the implant platform. The ΔMBL of tissue-level implants was 1.03 mm, which was slightly higher than 0.81 mm of bone-level implants, but there was no significant difference (p 0.05). No contributing factor associated with ΔMBL was identified by multivariate regression analysis in this study.Within the limits of this retrospective analysis, the ΔMBL of tissue-level implants is similar to that of bone-level implants after placed with simultaneous GBR, and both types of implants can achieve desirable marginal bone stability.

The prevalence of SARS-CoV-2 variants of concern (VOCs) is still escalating throughout the world. However, the level of neutralization of the inactivated viral vaccine recipients' sera and convalescent sera against all VOCs, including B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), B.1.617.2 (Delta), and B.1.1.529 (Omicron) remains to be lack of comparative analysis. Therefore, we constructed pseudoviruses of five VOCs using a lentiviral-based system and analyzed their viral infectivity and neutralization resistance to convalescent and BBIBP-CorV vaccinee serum at different times. Our results show that, compared with the wild-type strain (WT), five VOC pseudoviruses showed higher infection, of which B.1.617.2 and B.1.1.529 variant pseudoviruses exhibited higher infection rates than wild-type or other VOC strains, respectively. Sera from 10 vaccinated individuals at the 1, 3 and 5-month post second dose or from 10 convalescent at 14 and 200 days after discharge retained neutralizing activity against all strains but exhibited decreased neutralization activity significantly against the five VOC variant pseudoviruses over time compared to WT. Notably, 100% (30/30) of the vaccinee serum samples showed more than a 2.5-fold reduction in neutralizing activity against B.1.1.529, and 90% (18/20) of the convalescent serum samples showed more than 2.5-fold reduction in neutralization against B.1.1.529. These findings demonstrate the reduced protection against the VOCs in vaccinated and convalescent individuals over time, indicating that it is necessary to have a booster shot and develop new vaccines capable of eliciting broad neutralization antibodies.

Neurofibroma type 1 (NFI) is an autosomal dominant genetic disease associated with cancer and cognitive dysfunction. The 50% de novo mutation rate and numerous mutation types of the NF1 gene pose challenges to preimplantation genetic testing (PGT) for NFI. In 2020, a couple was subjected to next-generation sequencing (NGS)-based PGT at our center. Both husband and wife carried de novo NF1 mutations. Biopsied trophectoderm samples underwent whole-genome amplification using multiple displacement amplification. NGS was conducted to detect NF1 mutations as well as 105 closely linked single-nucleotide polymorphisms (SNPs) flanking NF1. Since no affected family member was available, the affected embryos identified by NF1-targeted sequencing were assigned as probands, based on which a haplotype analysis was constructed with informative SNP sites. Sanger sequencing was administrated for verification. Aneuploidy screening through NGS was performed in the same PGT cycle. Finally, among the nine biopsied blastocysts, two unaffected and euploid blastocysts were obtained in the couple, and one of the blastocysts was transferred in a frozen–thawed embryo transfer (FET) cycle. The outcome of FET is under observation. This study is the first to apply NGS-based PGT for NFI. In cases of sporadic NFI cases without affected family members, NGS-SNP can perform haploid analysis in an accurate manner by assigning the affected embryos as probands.

While widely used for ovulation induction in assisted reproductive technology, the clinical efficacy of letrozole for endometrial preparation prior to frozen-thawed embryo transfer (FET) cycles remains yet to be elucidated. We performed a meta-analysis to compare pregnancy outcomes after letrozole use with those of other endometrial preparation protocols in patients undergoing FET. PubMed, Scopus, Embase and the Cochrane Library were searched for eligible studies. Clinical pregnancy rate (CPR), live birth rate (LBR) and birth defect rate (BDR) were analysed using odds ratio (OR) and 95% confidence interval (CI). A total of 10 studies representing 75 968 FET cycles were included. Comparable CPR and LBR were observed when comparing letrozole administration with natural cycle (OR 1.24, 95% CI: 0.69 - 2.24; OR 1.18, 95% CI: 0.60 - 2.32), artificial cycle (OR 1.46, 95% CI: 0.87 - 2.44; OR 1.39, 95% CI: 0.77 - 2.52), and artificial cycle with gonadotropin-releasing hormone agonist suppression (OR 1.11, 95% CI: 0.78 - 1.59; OR 1.18, 95% CI: 0.82 - 1.68). Pooled results of the limited studies comparing letrozole with human menopausal gonadotropin demonstrated a similar CPR between groups (OR 1.46, 95% CI: 0.29 - 7.21, two studies), but the letrozole group had a statistically lower LBR (OR 0.67, 95% CI: 0.52 - 0.86, one study). No increased BDR was observed in the letrozole group compared to natural cycles or artificial cycles (OR 0.98, 95% CI: 0.60 - 1.61; OR 1.39, 95% CI; 0.84 - 2.28). This pooled analysis supports the use of letrozole as an efficacious and safe alternative to mainstream regimens for endometrial preparation in FET cycles.Obwohl Letrozol oft in der assistierten Reproduktionstechnik zur Einleitung der Ovulation eingesetzt wird, ist die klinische Wirksamkeit von Letrozol bei der Vorbereitung des Endometriums vor Transferzyklen von konservierten/aufgetauten Embryos (FET) noch unklar. Wir führten eine Metaanalyse durch, um das Schwangerschafts-Outcome nach dem Einsatz von Letrozol mit denen anderer Protokolle zur Vorbereitung des Endometriums bei Patientinnen, bei denen ein FET vorgenommen wurde, zu vergleichen. Die Datenbanken von PubMed, Scopus, Embase und the Cochrane Library wurden nach passenden Studien durchsucht. Die klinische Schwangerschaftsraten (SR), Lebendgeburtenraten (LGR) und Geburtsfehlerraten wurden mithilfe von Odds Ratio (OR) und 95%-Konfidenzintervallen (KI) analysiert. Insgesamt wurden 10 Studien, die 75 968 FET-Zyklen darstellten, in die Analyse aufgenommen. Die SR und die LGR für Letrozol waren statistisch vergleichbar mit den Werten für natürliche Zyklen (OR 1,24; 95%-KI 0,69 – 2,24 bzw. OR 1,18, 95%-KI 0,60 – 2,32), künstliche Zyklen (OR 1,46; 95%-KI 0,87 – 2,44 bzw. OR 1,39; 95%-KI 0,77 – 2,52), sowie künstliche Zyklen mit Gonadotropin-Releasing-Hormon-Agonisten-Suppression (OR 1,11; 95%-KI 0,78 – 1,59 bzw. OR 1,18; 95%-KI 0,82 – 1,68). Die gepoolten Ergebnisse der begrenzten Anzahl an Studien, die Letrozol mit humanem Menopausengonadotropin verglichen, zeigten eine ähnliche SR für beide Gruppen (OR 1,46; 95%-KI 0,29 – 7,21, 2 Studien), wobei die Letrozol-Gruppe eine statistisch niedrigere LGR aufwies (OR 0,67; 95%-KI 0,52 – 0,86, 1 Studie). Die Geburtsfehlerrate in der Letrozol-Gruppe war nicht erhöht verglichen mit den Geburtsfehlerraten für natürliche Zyklen bzw. künstliche Zyklen (OR 0,98; 95%-KI 0,60 – 1,61 bzw. OR 1,39; 95%-KI 0,84 – 2,28). Diese gepoolte Analyse weist darauf hin, dass Letrozol eine wirksame und sichere Alternative zu den Standardprotokollen bei der Vorbereitung des Endometriums in FET-Zyklen darstellen könnte.

DNA methylation has been implicated as an important mechanism for the development of uterine corpus endometrial carcinoma (UCEC), indicating that methylation-driven genes may be potential biomarkers for survival prediction. In this study, we aimed to identify a new prognostic methylation signature for UCEC based on differentially expressed genes (DEGs) and long noncoding RNAs (lncRNAs) (DELs). Sample-matched RNA-sequencing and methylation-array data were downloaded from The Cancer Genome Atlas database, by analysis of which a total of 269 DEGs and 4 DELs were identified to be methylation driven. Least absolute shrinkage and selection operator analysis screened that 14 methylation-driven genes were significantly associated with overall survival (OS) and thus were used as a signature to establish a prognostic risk model. Based on the median threshold, the patients were divided into the low-risk and the high-risk groups, which showed significantly different survival periods under the Kaplan-Meier curve. The area under receiver operating characteristic curve (AUC) was 0.934, 0.919, and 0.952 for the training, validation, and entire cohort, respectively. Stratification analysis showed that the established risk model may add prognostic values to conventional clinical factors (age, neoplasm histologic grade, and clinical stage). A nomogram was constructed based on the risk model and clinical parameters, with the AUC of 0.978 and c-index of 0.8079. Database for Annotation, Visualization, and Integrated Discovery (DAVID) function enrichment and Human Protein Atlas (HPA) protein expression validation showed 5 of these 14 genes may be especially important for UCEC (hypermethylated lowly expressed: CCBE1, FOXL2, PHLDB2, and DTNA; hypomethylated highly expressed: CCNE1). Comparison with breast cancer in the methylation level indicated ABCA12, CCNE1, and CLRN3 may be specific methylation-driven genes for UCEC. LncRNA HCG11 may function by coexpressing with DTNA. In conclusion, this 14-DNA methylation signature combined with clinical factors may a potentially effective biomarker in predicting OS for UCEC patients.

Uterine corpus endometrial carcinoma (UCEC) is one of the leading causes of death from gynecological cancer due to the high recurrence rate. A recent study indicated that molecular biomarkers can enhance the recurrence prediction power if they were integrated with clinical information. In this study, we attempted to identify a new multi-RNA-type-based molecular biomarker for predicting the recurrence risk and recurrence-free survival (RFS). Matched mRNA (including lncRNA) and miRNA RNA-sequencing data from 463 UCEC patients (

PURPOSE: To evaluate the efficacy of preimplantation genetic testing (PGT) for α- and β-double thalassemia combined with aneuploidy screening using next-generation sequencing (NGS). METHODS: An NGS-based PGT protocol was performed between 2017 and 2018 for twelve couples, each of which carried both α- and β-thalassemia mutations. Trophectoderm biopsy samples underwent whole-genome amplification using multiple displacement amplification (MDA), followed by NGS for thalassemia detection and aneuploidy screening. A selection of several informative single nucleotide polymorphisms (SNPs) established haplotypes. Aneuploidy screening was performed only on unaffected noncarriers and carriers. Unaffected and euploid embryos were transferred into the uterus through frozen-thawed embryo transfer (FET). RESULTS: A total of 280 oocytes were retrieved following 18 ovum pick-up (OPU) cycles, with 182 normally fertilized and 112 cultured to become blastocysts. One hundred and seven (95.5%, 107/112) blastocysts received conclusive PGT results, showing 56 (52.3%, 56/107) were unaffected. Thirty-seven (66.1%, 37/56) of the unaffected were also identified as euploid. One family had no transferable embryos. Unaffected and euploid embryos were then transferred into the uterus of the other 11 couples resulting in 11 healthy live births. The clinical pregnancy rate was 61.1% (11/18) per OPU and 68.8% (11/16) per FET, with no miscarriage reported. Seven families accepted the prenatal diagnosis and received consistent results with the NGS-based PGT. CONCLUSION: This study indicated that NGS could realize the simultaneous PGT of double thalassemia and aneuploidy screening in a reliable and accurate manner. Moreover, it eliminated the need for multiple biopsies, alleviating the potential damages to the pre-implanted blastocysts. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10815-020-01732-7) contains supplementary material, which is available to authorized users.

Backgroud & AimsCurrently, the COVID-19 pandemic, caused by SARS-CoV-2 infection, represents a serious public health problem worldwide. Although it has been shown that ACE2 serves as the main receptor for SARS-CoV-2 entry into host cells, studies have shown that ACE2 is expressed at extremely low levels in various tissues, especially in some organs where virus particles have been found, such as the heart and liver. Therefore, these organs potentially express additional SARS-CoV-2 receptors that have not yet been discovered.Methods & ResultsHere, by a genome-wide CRISPR-Cas9 activation library screening, we found that ASGR1 promoted SARS-CoV-2 infection of 293T cells. In Huh-7 and HepG2 cell lines, simultaneous knock out of ACE2 and ASGR1 prevented SARS-CoV-2 pseudovirus infection. In the immortalized THLE-2 hepatocyte cell line and primary liver parenchymal cells, both of which hardly express ACE2, SARS-CoV-2 could successfully establish an infection. After treatment with ASGR1 antibody, the infection rate significantly reduced. This suggests that SARS-CoV-2 infects liver cells mainly through an ASGR1-dependent mechanism. Finally, we also found that the soluble ASGR1 could not only prevent the SARS-CoV-2 pseudovirus, which binds to the ASGR1 receptors, from infecting host liver cells, but also had a protective effect on those expressing ACE2, indicating that administration of soluble ASGR1 protein may represent a new treatment approach.ConclusionsColletively, these findings indicate that ASGR1 is a candidate receptor for SARS-CoV-2 that promotes infection of liver cells.Lay SummaryWe show that ASGR1 is a candidate receptor for SARS-CoV-2 to infect liver cells.

HIV-specific chimeric antigen receptor (CAR) T cells have been developed to target latently infected CD4+ T cells that express virus either spontaneously or after intentional latency reversal. However, the T-cell exhaustion and the patient-specific autologous paradigm of CAR-T hurdled the clinical application. Here, we created HIV-specific CAR-T cells using human peripheral blood mononuclear cells and a 3BNC117-E27 CAR (3BE CAR) construct that enables the expression of PD-1 blocking scFv E27 and the single-chain variable fragment of the HIV-1-specific broadly neutralizing antibody 3BNC117 to target native HIV envelope glycoprotein (Env). In comparison with T cells expressing 3BNC117-CAR alone, 3BE CAR-T cells showed greater anti-HIV potency with stronger proliferation capability, higher killing efficiency (up to ~75%) and enhanced cytokine secretion in the presence of HIV envelope glycoprotein-expressing cells. Furthermore, our approach achieved high levels (over 97%) of the TCR-deficient 3BE CAR-T cells with the functional inactivation of endogenous TCR to avoid graft-versus-host disease without compromising their antiviral activity relative to standard anti-HIV CAR-T cells. These data suggest that we have provided a feasible approach to large-scale generation of “off-the-shelf” anti-HIV CAR-T cells in combination with antibody therapy of PD-1 blockade, which can be a powerful therapeutic candidate for the functional cure of HIV.

The SARS-CoV-2 variants B.1.617.1 (Kappa) contain multiple mutations in the spike protein. However, the effect of B.1.617.1 lineage-related mutants on viral infectivity and inactivated-virus vaccine efficacy remains to be defined. We therefore constructed 12 B.1.617.1-related pseudoviruses and systematically studied the effects of mutations on virus infectivity and neutralization resistance to convalescent and inactivated virus vaccine sera. Our results show that the B.1.617.1 variant exhibited both higher infectivity and neutralization resistance in sera at 1 or 3 months after vaccination of 28 individuals and at 14 and 200 days after discharge of 15 convalescents. Notably, 89% of vaccines and 100% of the convalescent serum samples showed more than 2.5-fold reduction in neutralization against one single mutation: E484Q. Besides, we found a significant decrease in neutralizing activity in convalescent patients and BBIBP-CorV vaccines for B.1.1.529. These findings demonstrate that inactivated-virus vaccination or convalescent sera showed reduced, but still significant, neutralization against the B.1.617.1 variant.

Background In preimplantation genetic testing for aneuploidy (PGT-A), appropriate evaluation of mosaic embryos is important because of the adverse implications of transferring embryos with high-level mosaicism or discarding those with low-level mosaicism. Despite the availability of multiple reliable techniques for PGT-A, data comparing the detection of mosaicism using these techniques are scarce. To address this gap in the literature, we compared the detection ability of the two most commonly used PGT-A platforms, next-generation sequencing (NGS) and the single-nucleotide polymorphism (SNP) array, for mosaic embryos. Results We retrospectively reviewed the data of PGT-A or preimplantation genetic testing for chromosomal structural rearrangements (PGT-SR) conducted at our center from January 2018 to October 2020, and selected blastocysts that underwent aneuploidy screening with both an SNP array and NGS. Trophectoderm biopsy, multiple displacement amplification (MDA), and aneuploidy screening with an SNP array were conducted on the enrolled blastocysts. When the SNP array indicated mosaicism, NGS was performed on the corresponding MDA product for verification. Among the 105 blastocysts diagnosed with mosaicism with the SNP array, 80 (76.19%) showed mosaicism in NGS, with complete and partial concordance rates of 47.62% (50/105) and 18.10% (19/105), respectively. The complete discordance rate of the two platforms was 34.29% (36/105). That is, 10.48% (11/105) of the blastocysts were diagnosed with completely different types of mosaicism with the two platforms, while 13.33% (14/105) and 10.48% (11/105) of the embryos diagnosed as showing mosaicism with SNP were detected as showing aneuploidy and euploidy with NGS, respectively. Conclusions The consistency of NGS and the SNP array in the diagnosis of embryo mosaicism is extremely low, indicating the need for larger and well-designed studies to determine which platform is more accurate in detecting mosaic embryos.

Adoptive cellular immunotherapy therapy using broadly neutralizing antibody-based chimeric antigen receptor-T cells (bNAb-based CAR-T) has shown great potency and safety for the functional cure of HIV. The efficacy of bNAb-based CAR-T cells could be compromised by adaptive resistance during HIV chronic infection according to the phenomenon that cellular exhaustion was observed in endogenous cytotoxic T-lymphocytes (CTLs) along with upregulated expression of PD−1. Here, we created HIV-specific CAR-T cells using human peripheral blood mononuclear cells (PBMCs) and a 3BNC117-DNR CAR (3BD CAR) construct that enables the expression of PD-1 dominant negative receptor (DNR) and the single-chain variable fragment of the HIV-1-specific broadly neutralizing antibody 3BNC117 to target native HIV envelope glycoprotein (Env). Compared with HIV CAR expression alone, 3BD CAR-T cells displayed potent lytic and functional responses to Env-expressing cell lines and HIV-infected CD4+T cells. Moreover, 3BD CAR-T cells can kill HIV-latently-infected cell lines, which are reactivated by the secretory cytokines of effector cells followed by contact with initial HIV-expressing fraction. Furthermore, bioluminescence imaging indicated that 3BD CAR-T cells displayed superior anti-HIV function in an HIV NCG mouse model of transplanting Env+/PD-L1+cells (LEL6). These studies suggested that our proposed combinational strategy of HIV CAR-T therapy with PD-1 blockade therapy is feasible and potent, making it a promising therapeutic candidate for HIV functional cure.

Human immunodeficiency virus type 1 (HIV-1) cannot be completely eliminated because of existence of the latent HIV-1 reservoir. However, the facts of HIV-1 latency, including its establishment and maintenance, are incomplete. FKBP3, encoded by the FKBP3 gene, belongs to the immunophilin family of proteins and is involved in immunoregulation and such cellular processes as protein folding. In a previous study, we found that FKBP3 may be related to HIV-1 latency using CRISPR screening. In this study, we knocked out the FKBP3 gene in multiple latently infected cell lines to promote latent HIV-1 activation. We found that FKBP3 could indirectly bind to the HIV-1 long terminal repeat through interaction with YY1, thereby recruiting histone deacetylase 1/2 to it. This promotes histone deacetylation and induces HIV-1 latency. Finally, in a primary latent cell model, we confirmed the effect of FKBP3 knockout on the latent activation of HIV-1. Our results suggest a new mechanism for the epigenetic regulation of HIV-1 latency and a new potential target for activating latent HIV-1. IMPORTANCE The primary reason why AIDS cannot be completely cured is the existence of a latent HIV-1 reservoir. Currently, the facts of HIV-1 latency, including its establishment and maintenance, are incomplete. Using a CRISPR library in our earlier screening of genes related to HIV-1 latency, we identified FBKP3 as a candidate gene related to HIV-1 latency. Therefore, in this mechanistic study, we first confirmed the HIV-1 latency-promoting effect of FKBP3 and determined that FKBP3 promotes histone deacetylation by recruiting histone deacetylase 1/2 to the HIV-1 long terminal repeat. We also confirmed, for the first time, that FKBP3 can act as a transcription factor (TF) recruitment scaffold and participate in epigenetic regulation of HIV-1 latency. These findings suggest a new mechanism for the epigenetic regulation of HIV-1 latency and a new potential target for activating latent HIV-1.

Previous observational studies have shown that exposure to ambient temperature and air pollution were associated with the incidence of gestational diabetes mellitus (GDM). However, the susceptible time window of non-optimal temperature on GDM is still unknown, and the interaction with air pollution has not been examined. We conducted a prospective cohort study in Guangzhou, China to investigate the windows of susceptibility of temperature extremes and variability on the risk of GDM and to explore any interaction effect with air pollution. Daily maximum (Tmax), minimum temperature (Tmin) and diurnal temperature range (DTR) were obtained from Guangdong Meteorological Service. Distributed lag non-linear models with a logistic regression were applied to assess the effect of temperature extremes and DTR in different weeks of gestation on GDM. To examine the interaction effect, relative excess risk due to interaction index, attributable proportion and synergy index were calculated. There were 5,165 pregnant women enrolled, of which 604 were diagnosed with GDM (11.7%). Compared with a reference temperature (50th percentile of Tmax), we found that extreme high temperature (99th percentile of Tmax) exposure during 21st and 22nd gestational weeks was associated with an increased risk of GDM. Extreme low temperature (1st percentile of Tmax) exposure during 14th to 17th weeks increased the risk of GDM. We observed that per 1 °C increment of DTR during 21st to 24th weeks was associated with an elevated GDM risk. No interaction effect of temperature extremes or variability with air pollution on GDM were observed. Our results suggested that non-optimal temperature is an independent risk factor of GDM. The time window of susceptibility for extreme temperatures and DTR exposure on the risk of GDM generally occurred in second trimester of pregnancy. In the context of climate change, our study has important implications for reproductive health and justifies more research in different climate zones.