A Virus-Inspired Inhalable Liponanogel Induces Potent Antitumor Immunity and Regression in Metastatic Lung Tumors.

Pulmonary delivery of immunostimulatory agents such as poly(I:C) to activate double-stranded RNA sensors MDA5 and RIG-I within lung-resident antigen-presenting cells is a potential strategy to enhance antitumor immunity by promoting type I interferon secretion. However, following pulmonary delivery, poly(I:C) suffers from rapid degradation and poor endosomal escape, thus limiting its potency. Inspired by the structure of a virus that utilizes internal viral proteins to tune the loading and cytosolic delivery of viral nucleic acids, we developed a liponanogel (LNG)-based platform to overcome the delivery challenges of poly(I:C). The LNG consisted of an anionic polymer hyaluronic acid-based nanogel core coated by a lipid shell, which served as a protective layer to stabilize the nanogel core in the lungs. The nanogel core was protonated within acidic endosomes to enhance the endosomal membrane permeability and cytosolic delivery of poly(I:C). After pulmonary delivery, LNG-poly(I:C) induced 13.7-fold more IFNß than poly(I:C) alone and 2-fold more than poly(I:C) loaded in the state-of-art lipid nanoparticles (LNP-poly(I:C)). Moreover, LNG-poly(I:C) induced more potent CD8+ T cell immunity and stronger therapeutic effects than LNP-poly(I:C). The combination of LNG-poly(I:C) and PD-L1 targeting led to regression of established lung metastases. Due to the ease of manufacturing and the high biocompatibility of LNG, pulmonary delivery of LNG may be broadly applicable to the treatment of different lung tumors and may spur the development of innovative strategies for cancer immunotherapy.

Predicting lung cancer survival prognosis based on the conditional survival bayesian network.

Lung cancer is a leading cause of cancer deaths and imposes an enormous economic burden on patients. It is important to develop an accurate risk assessment model to determine the appropriate treatment for patients after an initial lung cancer diagnosis. The Cox proportional hazards model is mainly employed in survival analysis. However, real-world medical data are usually incomplete, posing a great challenge to the application of this model. Commonly used imputation methods cannot achieve sufficient accuracy when data are missing, so we investigated novel methods for the development of clinical prediction models. In this article, we present a novel model for survival prediction in missing scenarios. We collected data from 5,240 patients diagnosed with lung cancer at the Weihai Municipal Hospital, China. Then, we applied a joint model that combined a BN and a Cox model to predict mortality risk in individual patients with lung cancer. The established prognostic model achieved good predictive performance in discrimination and calibration. We showed that combining the BN with the Cox proportional hazards model is highly beneficial and provides a more efficient tool for risk prediction.

Anti-thrombotic Effects Mediated by a Novel Dual-Target Peptide Inhibiting Both Platelet Aggregation and Thrombin Activity without Causing Bleeding.

BACKGROUND: Classical anticoagulants and antiplatelets are associated with high frequencies of bleeding complications or treatment failure when used as single agents. Thrombin plays an important role in the blood coagulation system. GP IIb/IIIa is the central receptor of platelets, which can recognize the Arg-Gly-Asp (RGD) sequence and activate platelets. MATERIAL AND METHODS: Molecular simulation and homology modeling were performed to design a novel dual-target anticoagulant short peptide (PTIP ). The activities of PTIP on coagulation and platelet in vitro were analyzed. The antithrombotic activity of PTIP was determined by pulmonary thromboembolism model, ferric chloride injury model and arteriovenous bypass thrombosis model. Bleeding effect and toxicity of PTIP were evaluated. RESULTS: We have constructed a novel dual-target peptide (PTIP) based on the direct thrombin inhibitor peptide (DTIP). PTIP was expressed at high levels in Pichia pastoris. PTIP interfered with thrombin-mediated coagulation and ADP-induced platelet aggregation in vitro. When injected intravenously or subcutaneously, PTIP showed potent and dose-dependent extension of aPTT and PT which were similar to DTIP; but only PTIP was capable of inhibiting platelet aggregation. PTIP (1.0 mg/kg) decelerated thrombosis formation in venous and arterial vessels induced by FeCl3 injury. PTIP (1.0 mg/kg) also prevented deep venous thrombosis and increased the survival rate associated with pulmonary thromboembolism. And PTIP effectively reduced thrombus length in arteriovenous bypass thrombosis model. Moreover, the antithrombotic dose of PTIP could not induce bleeding. CONCLUSION: These data establish that PTIP represents a novel antithrombotic agent whose effects involve both inhibition of platelet activation and reduction of fibrin generation. And PTIP not only can be used in venous thrombosis and arterial thrombosis, it can also replace the combined treatment of antiplatelet and anticoagulant drugs in thrombotic diseases.

Long-Term Effectiveness of Ear Molding and Factors Affecting Outcomes.

BACKGROUND: The EarWell System offers a correction opportunity for infants born with ear anomalies. However, the long-term effectiveness of ear molding remains unclear. This study aimed to explore the long-term effectiveness of this novel technique and to determine the risk factors for recurrence. METHODS: This retrospective, population-based cohort study was performed from 2017 through 2021. Infants who completed ear molding therapy and were followed up for longer than 6 months were enrolled. The main outcomes were immediate and long-term efficacy, which were graded by two blinded plastic surgeons. RESULTS: A total of 226 infants (334 ears) were recruited. The most common anomalies were helical deformities [113 ears (33.8%)], and the rarest were cryptotia [five ears (1.5%)] and conchal crus [five ears (1.5%)]. The age at initiation of treatment was a factor affecting both immediate ( P = 0.004) and long-term effectiveness ( P = 0.009). The type of anomaly also influenced long-term molding outcomes. For cup ears, the success rate of long-term outcomes (76.0%) was significantly lower than that of immediate outcomes (98.7%) ( P < 0.001). Prominent ear, cup ear, and microtia were found to be the most likely to relapse during long-term follow-up. The results of logistic regression also demonstrated age, duration time, and the type of anomaly to be risk factors of ear molding effects. CONCLUSIONS: The EarWell System was shown to be a secure and effective method for treatment of congenital ear anomalies. Some infants' ear anomalies recurred after successful immediate results. The age at initiation of treatment and the type of anomaly were predictors of long-term outcomes. CLINICAL QUESTION/LEVEL OF EVIDENCE: Risk, III.

Opportunistic Screening With Low-Dose Computed Tomography and Lung Cancer Mortality in China.

Importance: Despite the recommendations of lung cancer screening guidelines and the evidence supporting the effectiveness of population-based lung screening, a common barrier to effective lung cancer screening is that the participation rates of low-dose computed tomography (LDCT) screening among individuals with the highest risk are not large. There are limited data from clinical practice regarding whether opportunistic LDCT screening is associated with reduced lung-cancer mortality. Objective: To evaluate whether opportunistic LDCT screening is associated with improved prognosis among adults with lung cancer in mainland China. Design, Setting, and Participants: This cohort study included patients diagnosed with lung cancer at Weihai Municipal Hospital Healthcare Group, Weihai City, China, from 2016 to 2021. Data were analyzed from January 2022 to February 2023. Exposures: Data collected included demographic indicators, tumor characteristics, comorbidities, blood indexes, and treatment information. Patients were classified into screened and nonscreened groups on the basis of whether or not their lung cancer diagnosis occurred through opportunistic screening. Main Outcomes and Measures: Follow-up outcome indicators included lung cancer-specific mortality and all-cause mortality. Propensity score matching (PSM) was adopted to account for potential imbalanced factors between groups. The associations between LDCT screening and outcomes were analyzed using Cox regression models based on the matched data. Propensity score regression adjustment and inverse probability treatment weighting were used for sensitivity analysis. Results: A total of 5234 patients (mean [SD] baseline age, 61.8 [9.8] years; 2518 [48.1%] female) with complete opportunistic screening information were included in the analytical sample, with 2251 patients (42.91%) receiving their lung cancer diagnosis through opportunistic screening. After 1:1 PSM, 2788 patients (1394 in each group) were finally included. The baseline characteristics of the matched patients were balanced between groups. Opportunistic screening with LDCT was associated with a 49% lower risk of lung cancer death (HR, 0.51; 95% CI, 0.42-0.62) and 46% lower risk of all-cause death (HR, 0.54; 95% CI, 0.45-0.64). Conclusions and Relevance: In this cohort study of patients with lung cancer, opportunistic lung cancer screening with LDCT was associated with lower lung cancer mortality and all-cause mortality. These findings suggest that opportunistic screening is an important supplement to population screening to improve prognosis of adults with lung cancer.

A novel recombinant human microplasminogen induced complete posterior vitreous detachment without morphological change of retina in juvenile rabbits.

Vitreomacular traction syndrome results from persistent vitreoretinal adhesions in the setting of partial posterior vitreous detachment (PVD). Vitrectomy and reattachment of retina is an effective therapeutic approach. The adhesion between vitreous cortex and internal limiting membrane (ILM) of the retina is stronger in youth, which brings difficulties to induce PVD in vitrectomy. Several clinical investigations demonstrated that intravitreous injection of plasmin before vitrectomy could reduce the risk of detachment. In our study, a novel recombinant human microplasminogen (rhµPlg) was expressed by Pichia pastoris. Molecular docking showed that the binding of rhµPlg with tissue plasminogen activator (t-PA) was similar to plasminogen, suggesting rh µPlg could be activated by t-PA to generate microplasmin (µPlm). Moreover, rhµPlg had higher catalytic activity than plasminogen in amidolytic assays. Complete PVD was found at vitreous posterior pole of 125 µg rhµPlg-treated eyes without morphological change of retina in juvenile rabbits via intraocular injection. Our results demonstrate that rhµPlg has a potential value in the treatment of vitreoretinopathy.

Differentiation of true progression from treatment response in high-grade glioma treated with chemoradiation: a comparison study of 3D-APTW and 3D-PcASL imaging and DWI.

PURPOSE: To assess and compare the diagnostic performance of 3D amide proton-transfer-weighted (3D-APTW) imaging, 3D pseudocontinuous arterial spin-labeling (3D-PcASL) imaging, and diffusion-weighted imaging in distinguishing true progression (TP) from treatment response (TR) in posttreatment malignant glioma patients. MATERIALS AND METHODS: Forty-eight patients with suspected tumor recurrence were prospectively enrolled. Histological or longitudinal routine MRI follow-up over six months was assessed to confirm lesion type. The apparent diffusion coefficient (ADC), relative APTWmax (rAPTW), and relative CBFmax values (rCBF) were measured in lesions with enhancing regions on post-gadolinium T1 -weighted MRI. MRI parameters between the TP and TR groups were compared using Student's t tests. In addition, a receiver operating characteristic (ROC) curve was constructed, and the area under the ROC curve (AUC) was calculated to assess the differentiation diagnostic performance of each parameter. RESULTS: The TP group showed a significantly higher rAPTW and rCBF than the TR group; the AUCs of rAPTW and rCBF to distinguish between TP and TR were 0.911 (with sensitivity of 90.3% and specificity of 82.4%) and 0.852 (with sensitivity of 80.6% and specificity of 82.4%), respectively. By adding the rAPTW values to rCBF values, the diagnostic ability was improved from 0.852 to 0.951. ADC showed no significant differences between the TP and TR groups, with an AUC lower than 0.70. CONCLUSION: Both 3D-PcASL and 3D-APTW imaging could distinguish TP from TR, and 3D-APTW had a better diagnostic performance. Combining the rAPTW values and rCBF values achieved a better diagnostic performance.

3D Amide Proton Transfer-Weighted Imaging for Grading Glioma and Correlating IDH Mutation Status: Added Value to 3D Pseudocontinuous Arterial Spin Labelling Perfusion.

PURPOSE: The goal of this study was to evaluate the diagnostic performance of 3D amide proton transfer-weighted (3D-APTW) imaging and 3D pseudocontinuous arterial spin labelling (3D-pCASL) alone and in combination in grading gliomas (low-grade glioma (LGG) vs. high-grade glioma (HGG)) and correlating isocitrate dehydrogenase (IDH) mutation status. PROCEDURES: Preoperatively, 81 patients with pathologically confirmed gliomas underwent 3.0-T magnetic resonance imaging (MRI) examinations. The APTW, relative APTW (rAPTW), cerebral blood flow (CBF), and relative CBF (rCBF) values were calculated to evaluate the solid components of the tumours. The MRI parameters were compared in the classification of gliomas by independent- and paired-samples t tests. A receiver operating characteristic (ROC) curve was constructed, and the area under the ROC curve (AUC) was calculated to assess the diagnostic performance of each parameter and the combination of the rAPTW and rCBF values. RESULTS: Patients with HGG showed significantly higher APTW, rAPTW, CBF, and rCBF values than those with LGG (all p < 0.001). In the ROC curve analysis, the AUC of rAPTW was the highest at 0.90. By adding the rAPTW signal to the rCBF values, the diagnostic ability of the combined parameters improved from 0.90 to 0.96. The rAPTW value yielded the highest AUC (0.92) in correlating the IDH mutation status, and the diagnostic ability improved to 0.96 by adding it to the rCBF value. CONCLUSION: 3D-APTW imaging combined with 3D-pCASL imaging may be used to aid assessment of grading glioma and IDH mutation status.

Design and fabrication of r-hirudin loaded dissolving microneedle patch for minimally invasive and long-term treatment of thromboembolic disease.

Cardiovascular disease is the leading cause of global mortality, with anticoagulant therapy being the main prevention and treatment strategy. Recombinant hirudin (r-hirudin) is a direct thrombin inhibitor that can potentially prevent thrombosis via subcutaneous (SC) and intravenous (IV) administration, but there is a risk of haemorrhage via SC and IV. Thus, microneedle (MN) provides painless and sanitary alternatives to syringes and oral administration. However, the current technological process for the micro mould is complicated and expensive. The micro mould obtained via three-dimensional (3D) printing is expected to save time and cost, as well as provide a diverse range of MNs. Therefore, we explored a method for MNs array model production based on 3D printing and translate it to micro mould that can be used for fabrication of dissolving MNs patch. The results show that r-hirudin-loaded and hyaluronic acid (HA)-based MNs can achieve transdermal drug delivery and exhibit significant potential in the prevention of thromboembolic disease without bleeding in animal models. These results indicate that based on 3D printing technology, MNs combined with r-hirudin are expected to achieve diverse customizable MNs and thus realize personalized transdermal anticoagulant delivery for minimally invasive and long-term treatment of thrombotic disease.

LTM-TCM: A comprehensive database for the linking of Traditional Chinese Medicine with modern medicine at molecular and phenotypic levels.

Benefiting from the development of network pharmacology, Traditional Chinese Medicine (TCM) shows great potential in modern drug discovery. Recently, more and more TCM-related databases have been established for both academic and industry research, but they are still insufficient in data standardization, integrity, and precision. To better accelerate the TCM research and overcome these shortcomings, we construct a web-based TCM platform, LTM-TCM, which is currently the most comprehensive TCM database that includes the following advantages: (1) High-quality data integration from fourteen TCM authoritative databases, especially with additional manual collected 41,025 clinical treatment records and 213 ancient Chinese medical books. (2) Accurate correction of multi-source TCM interactions (between symptoms, prescriptions, herbs, ingredients and targets) through in-house Biomedical Natural Language Processing (BioNLP) approaches in more than 30 million articles. (3) Diverse cross-field pipelines (e.g., bioactive ingredients screening, targets prediction, and mechanism prediction, etc.) help integrating traditional medicine with modern science in common aspects at both the molecular and phenotypic levels. In summary, LTM-TCM contains 1928 symptoms, 48,126 prescriptions, 9122 plants, 34,967 ingredients, 13,109 targets and 1170,133 interactions among all TCM related components. LTM-TCM has both Chinese and English interfaces, and it is accessible at http://cloud.tasly.com/#/tcm/home.

A novel oncotherapy strategy: Direct thrombin inhibitors suppress progression, dissemination and spontaneous metastasis in non-small cell lung cancer.

BACKGROUND AND PURPOSE: Cancer cachexia and cancer-associated thrombosis are potentially fatal outcomes of advanced cancer. Nevertheless, thrombin expression in non-small cell lung cancer (NSCLC) primary tumour tissues and the association between prognosis of NSCLC patients remain largely unknown. EXPERIMENTAL APPROACH: Clinical pathological analysis was performed to determine the relationship between thrombin and tumour progression. Effects of r-hirudin and direct thrombin inhibitor peptide (DTIP) on cancer progression were evaluated. Western blotting, immunohistochemistry, and immunofluorescence were used to explore the inhibition mechanism of r-hirudin and DTIP. The therapeutic effect of the combination of DTIP and chemotherapy was determined. KEY RESULTS: Thrombin expression in NSCLC tissues was closely related to clinicopathological features and the prognosis of patients. Thrombin deficiency inhibited tumour progression. The novel thrombin inhibitors, r-hirudin and DTIP, inhibited cell invasion and metastasis in vitro. They inhibited tumour growth and metastasis in orthotopic lung cancer model, inhibited cell invasion, and prolonged survival after injection of tumour cells via the tail vein. They also inhibited angiogenesis and spontaneous metastases from subcutaneously inoculated tumours. The promotion by thrombin of invasion and metastasis was abolished in PAR-1-deficient NSCLC cells. r-hirudin and DTIP inhibited tumour progression through the thrombin-PAR-1-mediated RhoA and NF-κB signalling cascades via inhibiting MMP9 and IL6 expression. DTIP potentiated chemotherapy-induced growth and metastatic inhibition and inhibited chemotherapy-induced resistance in mice. CONCLUSIONS AND IMPLICATIONS: Thrombin makes a substantial contribution, together with PAR-1, to NSCLC malignancy. The anti-coagulants, r-hirudin and DTIP, could be used in anti-tumour therapy and a combination of DTIP and chemotherapy might improve therapeutic effects.

Reconstruction of Distal Nasal Defects With a Large Postauricular Skin-Fat-Fascia Composite Graft.

BACKGROUND: Composite grafts have previously been reported to achieve a good outcome for nasal defect repair, but composite grafts have greater metabolic needs than simple skin. Therefore, the traditionally recommended size of a composite graft for nasal reconstruction is less than 1.5 cm in diameter. However, the distal nose is generally well supplied with blood vessels, which might support the use of larger composite grafts in such a highly vascularized recipient site. The aim of the article is to investigate whether a large skin-fat-fascia composite graft (larger than 2.0 cm) is viable for the repair of partial-thickness nasal defects. METHODS: From October 2017 to December 2019, 13 patients with partial-thickness nasal defects underwent nasal reconstruction using a large postauricular skin-fat-fascia composite graft. Cases were followed up for 3 to 14 months postoperatively. The aesthetic outcome was evaluated in comparison with preoperative digital images. RESULTS: Skin-fat-fascia composite grafts survived without graft necrosis, dermal fibrosis, or skin contraction in all cases. Favorable aesthetic outcomes were obtained in all patients, and no further revision surgery was need. CONCLUSIONS: A postauricular composite graft larger than 2.0 cm is a safe and effective reconstruction approach for partial-thickness nasal defects. This technique offers significant advantages in terms of no additional facial scar, no visible asymmetry on the face, no additional surgery for revision, and with mild scar in the donor site of the postauricular region.

Early argatroban and antiplatelet combination therapy in acute non-lacunar single subcortical infarct associated with mild intracranial atherosclerosis.

BACKGROUND: Patients with acute non-lacunar single subcortical infarct (SSI) associated with mild intracranial atherosclerosis (ICAS) have a relatively high incidence of early neurological deterioration (END), resulting in unfavorable functional outcomes. Whether the early administration of argatroban and aspirin or clopidogrel within 6-12 h after symptom onset is effective and safe in these patients is unknown. METHODS: A review of the stroke database of Weihai Municipal Hospital, Cheeloo College of Medicine, Shandong University and Qingdao Center Hospital, Qingdao University Medical College in China was undertaken from May 2017 to January 2020 to identify all patients with non-lacunar SSI caused by ICAS within 6-12 h of symptom onset based on MRI screening. Patients were divided into two groups, one comprising those who received argatroban and mono antiplatelet therapy with aspirin or clopidogrel on admission (argatroban group), and the other those who received dual antiplatelet therapy (DAPT) with aspirin and clopidogrel during hospitalization (DAPT group). The primary outcome was recovery by 90 days after stroke based on a modified Rankin scale (mRS) score (0 to 1). The secondary outcome was END incidence within 120 h of admission. Safety outcomes were intracranial hemorrhage (ICH) and major extracranial bleeding. The probability of clinical benefit (mRS score 0-1 at 90 days) was estimated using multivariable logistic regression analysis. RESULTS: A total of 304 acute non-lacunar SSI associated with mild ICAS patients were analyzed. At 90 days, 101 (74.2%) patients in the argatroban group and 80 (47.6%) in the DAPT group had an mRS score that improved from 0 to 1 (P < 0.001). The relative risk (95% credible interval) for an mRS score improving from 0 to 1 in the argatroban group was 1.50 (1.05-2.70). END occurred in 10 (7.3%) patients in the argatroban group compared with 37 (22.0%) in the DAPT group (P < 0.001). No patients experienced symptomatic hemorrhagic transformation. CONCLUSIONS: Early combined administration of argatroban and an antiplatelet agent (aspirin or clopidogrel) may be beneficial for patients with non-lacunar SSI associated with mild ICAS identified by MRI screening and may attenuate progressive neurological deficits. TRIAL REGISTRATION: Our study is a retrospectively registered trial.

Network bioinformatics analysis provides insight into drug repurposing for COVID-19.

The COVID-19 disease caused by the SARS-CoV-2 virus is a health crisis worldwide. While developing novel drugs and vaccines is long, repurposing existing drugs against COVID-19 can yield treatments with known preclinical, pharmacokinetic, pharmacodynamic, and toxicity profiles, which can rapidly enter clinical trials. In this study, we present a novel network-based drug repurposing platform to identify candidates for the treatment of COVID-19. At the time of the initial outbreak, knowledge about SARS-CoV-2 was lacking, but based on its similarity with other viruses, we sought to identify repurposing candidates to be tested rapidly at the clinical or preclinical levels. We first analyzed the genome sequence of SARS-CoV-2 and confirmed SARS as the closest virus by genome similarity, followed by MERS and other human coronaviruses. Using text mining and database searches, we obtained 34 COVID-19-related genes to seed the construction of a molecular network where our module detection and drug prioritization algorithms identified 24 disease-related human pathways, five modules, and 78 drugs to repurpose. Based on clinical knowledge, we re-prioritized 30 potentially repurposable drugs against COVID-19 (including pseudoephedrine, andrographolide, chloroquine, abacavir, and thalidomide). Our work shows how in silico repurposing analyses can yield testable candidates to accelerate the response to novel disease outbreaks.

Assembly of Nanofluidic MXene Fibers with Enhanced Ionic Transport and Capacitive Charge Storage by Flake Orientation.

MXenes are an emerging class of highly conductive two-dimensional (2D) materials with electrochemical storage features. Oriented macroscopic Ti3C2Tx fibers can be fabricated from a colloidal 2D nematic phase dispersion. The layered conductive Ti3C2Tx fibers are ideal candidates for constructing high-speed ionic transport channels to enhance the electrochemical capacitive charge storage performance. In this work, we assemble Ti3C2Tx fibers with a high degree of flake orientation by a wet spinning process with controlled spinning speeds and morphology of the spinneret. In addition to the effects of cross-linking of magnesium ions between Ti3C2Tx flakes, the electronic conductivity and mechanical strength of the as-prepared fibers have been improved to 7200 S cm-1 and 118 MPa, respectively. The oriented Ti3C2Tx fibers present a volumetric capacitive charge storage capability of up to 1360 F cm-3 even in a Mg-ion based neutral electrolyte, with contributions from both nanofluidic ion transport and Mg-ion intercalation pseudocapacitance. The oriented 2D Ti3C2Tx driven nanofluidic channels with great electronic conductivity and mechanical strength endows the MXene fibers with attributes for serving as conductive ionic cables and active materials for fiber-type capacitive electrochemical energy storage, biosensors, and potentially biocompatible fibrillar tissues.

[Development of Gun-type Tube-Guide Device of Laser Fiber].

With the development of laryngeal microsurgery, the requirements for the flexibility and convenience of surgical instruments are increasing. The research on related instruments has important value for the clinical application of laryngeal microsurgery. We have redesigned a gun-type tube-guide device of laser fiber by comparing the shortcomings of existing laser fiber introducers. The innovation of this design lies in its rotating nut device with adjustable laser angle and pre-bent tip. The corresponding in vitro laryngeal model experiment can realize multi-angle rotation of the instrument in the laryngeal cavity, which greatly increases the scope of laser surgery. During the operation, the rotating nut can be directly adjusted to avoid repeatedly removing the instrument to adjust the angle, which greatly improves the practicability and simplicity of the operation, which is worthy of further clinical research and promotion.

Enhanced Efficiency of flySAM by Optimization of sgRNA Parameters in Drosophila.

The flySAM/CRISPRa system has recently emerged as a powerful tool for gain-of-function studies in Drosophila melanogaster This system includes Gal4/UAS-driven dCas9 activators and U6 promoter-controlled sgRNA. Having established dCas9 activators superior to other combinations, to further enhance the efficiency of the targeting activators we systematically optimized the parameters of the sgRNA. Interestingly, the most efficient sgRNAs were found to accumulate in the region from -150bp to -450bp upstream of the transcription start site (TSS), and the activation efficiency showed a strong positive correlation with the GC content of the sgRNA targeting sequence. In addition, the target region is dominant to the GC content, as sgRNAs targeting areas beyond -600bp from the TSS lose efficiency even when containing 75% GC. Surprisingly, when comparing the activities of sgRNAs targeting to either DNA strand, sgRNAs targeting to the non-template strand outperform those complementary to the template strand, both in cells and in vivo In summary, we define criteria for sgRNA design which will greatly facilitate the application of CRISPRa in gain-of-function studies.

Detection and Treatment of Persistent Mullerian Duct Syndrome With Transverse Testicular Ectopia.

The coexistence of persistent Mullerian duct syndrome (PMDS) with transverse testicular ectopia (TTE) is extremely rare. Due to a lack of distinctive clinical features in the early stages, PMDS coexists with TTE is usuallydiagnosed when patients are examined for other diseases,including cryptorchidism and inguinal hernia. We present a case of a 51-year-old man who presented with a recurrent left indirect inguinal hernia for 2 years and right congenital cryptorchidism. The patient was diagnosed as PMDS with TTE by preoperative magnetic resonance imaging and underwent laparoscopic resection of the right transverse ectopic testis and Mullerian duct residues.