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Abstract In comparison to an open field, greenhouses utilize much more pesticides. The non-occupational exposure risk caused by pesticide drift is unknown. In this study, within 8 months (from March 2018 to October 2018), air samples were collected from indoor and outdoor houses and public areas near greenhouses in vegetable growing areas (eggplant, leek, garlic, etc.), and qualitative and quantitative analyses of pesticides were carried out. Using a 95% confidence interval, six pesticides (acetamiprid, difenoconazole, thiazophos, isoprocarb, malathion, and pyridaben) were detected. The results of the safety assessment showed that the non-cancer exposure risk of single pesticides for all residents in the agricultural areas was within the acceptable range, and the excess lifetime cancer risk of all residents inhaling difenoconazole exceeded 1E−6, and the agricultural region urgently needs increased cancer regulatory scrutiny. But combined toxicity of six pesticides not evaluated due to lack of suitable data. Comparison with open field scenes, the results show that pesticide levels to airborne are lower in greenhouse regions.

The traditional organic index can be greatly affected in the areas with high thermal evolution degree whilst combination of REE for oil-source correlation will improve the results. In addition, Re-Os dating is a quantitative method of geochronology, but the significance of the test results is quite controversial. In this paper, REE geochemical analysis and Re-Os dating of bitumen in the Banjie paleo-oil reservoir is carried out. The REE distribution pattern of bitumen was compared with that of the main source rocks in the area using the method of grey correlation. Results show that the ΣREE of bitumen in the Banjie paleo-oil reservoir is low, and the fractionation of light and heavy REEs is obvious, showing a weak negative Ce anomaly and an obvious negative Eu anomaly, which indicates that the bitumen was formed in a reducing environment and has not experienced any strong weathering or leaching. The REE distribution mode belongs to the right-leaning mode of LREE enrichment. The REE distribution pattern curve is similar to that of Devonian source rocks in this area, indicating a genetic relationship. The results of Re-Os dating show that the Re-Os model age of bitumen in the Banjie paleo-oil reservoir ranges mainly from (203.6±1.1) Ma to (238.5±4.2) Ma, with an average of (224.8±3.3) Ma (n=7). The last factor which disturbed the Re-Os system of the bitumen is the process of bitumen generation. The model age represents the age of bitumen formation in the Banjie paleo-oil reservoir, when oil was cracked into gas.

The Nanpanjiang basin hosts the world’s second-largest concentration of Carlin-type gold deposits. To decipher the origin and evolution of hydrothermal fluid, this study conducted Sm–Nd dating, in-situ trace element, and C-O-Sr isotopic analyses on three types of calcite samples from the giant Lannigou gold deposit in the Nanpanjiang basin, SW China. The type-I calcite, intergrown with Au-bearing arsenian pyrite, has an Sm–Nd isochron age of 213 ± 7 Ma (MSWD = 0.81), indicating that gold mineralization occurred in Late Triassic. The type-II calcite, which coexists with high-maturity bitumens and cut through the main-stage gold orebodies, yields an Sm–Nd age of 188 ± 14 Ma (MSWD = 0.34), representing a post-ore hydrocarbon accumulation event. The type-I and type-II calcite samples have low REE contents (5.28–51.6 ppm) and exhibit MREE-enriched and LREE-/HREE-depleted patterns. Combined with their identical C-O-Sr isotopes, we suggest that hydrothermal fluids responsible for the precipitation of type-I and type-II calcite samples were derived from a mixed metamorphic fluid and meteoric water source. In contrast, the type-III calcite samples, associated with realgar and orpiment, have distinct Mn, Sr, and As contents, REE patterns, and C-O-Sr isotopic composition from the type-I and II calcites, suggestive of different fluid sources. Based on our and previously published data, we propose that the fluid evolution, gold mineralization, and hydrocarbon accumulation in the Nanpanjiang basin are closely related to the Indosinian and Yanshanian orogenies in South China.

The coexistence of numerous Mississippi Valley-type (MVT) Pb-Zn deposits and oil/gas reservoirs in the world demonstrates that a close genetic link between them. The northern Yunnan–Guizhou depression (NYGD) is a tectonic unit containing Pb–Zn deposits, paleo-oil reservoirs and shale gas. However, previous studies on the relationship between hydrocarbon accumulation and Pb–Zn mineralization have been ignored. The Maoping Pb–Zn deposit is a large-sized MVT deposit in the NYGD where a large amount of solid bitumen (i.e., a paleo-oil reservoir) occurs, and it is an ideal area to study the relationship between hydrocarbon accumulation and lead–zinc mineralization. In this paper, the bitumen and pyrite associated with bitumen from the Xujiazhai (discovered in this study) and Xiaocaoba paleo-oil reservoirs and the Maoping Pb–Zn deposit are researched. Geological observation has revealed that bitumen occurs in dissolution pores, fractures and intercrystalline pores in dolomite of the Upper Devonian and Carboniferous. The bitumen from Xujiazhai, Maoping and Xiaocaoba with high and similar Raman equivalent reflectance (RmcRo%) indicates they are in over-mature level and may be derived from the cracking of early-accumulated crude oils. The bitumen in the Xujiazhai paleo-oil reservoir and Maoping deposit has an abundant Pb and Zn content, indicating petroleum liquids may act as the transporting agents of metallogenic metal elements, carrying them to sites where mineralization may take place. The source rocks may have provided not only the oil for paleo-oil/gas reservoirs, but also a portion of ore metals for the Pb-Zn mineralization. The bacterial sulfate reduction (BSR) and thermochemical sulfate reduction (TSR) processes were recorded by the pyrite in the paleo-oil reservoir, which was consistent with the mechanism of reduced sulfur formation in the Maoping deposit. In addition, the large negative (−27.7‰ to −5.7‰) and positive (.9‰ to 19.2‰) δ34S values of the pyrite associated with the bitumen in the paleo-oil reservoir were similar to those of the sulfide in the Maoping deposit. We believe that the formation and evolution of the paleo-oil reservoirs are closely related to the metallogenic process of the Maoping Pb–Zn deposit, and the sulfides in them have the same sulfur source and formation mechanism as reduced sulfur. BSR phenomena could occur in prior to migration of the hydrocarbons into the reservoir or low mature oil stage in paleo-oil reservoir; a small amount of H2SBSR was combined with metal ions in ancient oil reservoirs and deposits to form early subhedral, xenomorphic granular, fine-grained strawberry aggregate pyrite and/or gelatinous sphalerite. The paleo-gas reservoir formed by the evolution of the paleo-oil reservoir in the main metallogenic period potentially participated in the mineralization; that is, organic gas acted as a reducing agent and transformed SO42− in the upper Devonian Zaige Formation gypsum strata on the periphery of the Maoping lead–zinc mining area into H2STSR through TSR, providing reduced sulfur and creating the environmental conditions for mineralization. During or after the transformation of the paleo-oil reservoir to a paleo-gas reservoir, the decoupling of metals and organic complexes may have provided the ore metals for mineralization. Massive sulfide precipitation may have occurred during or after the paleo-oil reservoir cracked into the paleo-gas reservoir.

In Qinglong ore field, the paleo-oil reservoir is found to be associated with antimony deposits, and they have a close genetic relationship. In this study, the aromatics geochemistry of paleo-oil reservoir bitumen was studied to further discuss the thermochemical sulfate reduction (TSR) reaction and the mechanism of antimony mineralization. A total of 124 aromatic compounds were identified by gas chromatography–mass spectrometry (GC–MS) analysis in bitumen samples, including abundant phenanthrene series, dibenzothiophene series, fluoranthene series, chrysene series, and a small number of fluorene series, naphthalene series, dibenzofuran series, biphenyl series, and triaromatic steroid series. Aromatic parameters such as trimethylnaphthalene index (TMNr), methylphenanthrene index (MPI), methylphenanthrene distribution fraction (MPDF, F1, and F2), methyldibenzothiophene parameter (MDR), C28TAS-20S/(20R + 20S), and benzofluoranthene/benzo[e]pyrene indicate that the Qinglong paleo-oil reservoir is in over maturity level. The abundance of phenanthrene and chrysene aromatic compounds and a small amount of naphthalene series, benzofluoranthene, fluoranthene, pyrene, anthracene, retene, perylene, and biphenyl suggest that the organic matter source of the paleo-oil reservoir was mainly low aquatic organisms, mixed with a small amount of higher plant. They detected a certain number of compounds, such as retene, triaromatic steroid series, and perylene, the ternary diagram of DBF–DBT–F and binary plot of Pr/Ph–DBT/P, DBT/(F + DBT)–DBF/(F + DBF), and Pr/Ph–DBT/DBF reveal that the source rock of the paleo-oil reservoir was formed in the marine environment of weak oxidation and weak reduction. The comprehensive analysis shows that the Qinglong paleo-oil reservoir originated from Devonian source rocks, just like other paleo-oil reservoirs and natural gas reservoirs in the Nanpanjiang basin. Abundant dibenzothiophene series were detected, indicating that the paleo-oil reservoir underwent a certain degree of TSR reaction. We believe that the gas reservoir formed by the evolution of the oil reservoir in the ore field participated in antimony mineralization; that is, hydrocarbon organic matter acted as a reducing agent and transformed SO42− in oilfield brine into H2S through TSR, providing reduced sulfur and creating environmental conditions for mineralization.

Abstract Aim Spinal cord stimulation (SCS) is an effective method to treat neuropathic pain. It is necessary to identify the responders of SCS analgesia before implantation. The aim of this study is to investigate the relationship between the cortical dynamics and SCS analgesia responders in pain management. Methods Resting‐state EEG recording was performed in patients who underwent short‐term implantation of spinal cord stimulation for pain therapy. We then did spectral analysis to capture the pattern of cortical oscillation between neuromodulation therapy analgesia responders and nonresponders. Results About 58.3% (14 out of 24) of participants were considered as analgesia responders, with average visual analogue scores reduction of 4.8 ± 1.0 after surgery, and 2.1 ± 0.7 for the nonresponder subgroup, respectively. The alpha oscillation was significantly enhanced in responder cohort compared with nonresponders. We also observed an increasing spectral power of gamma band in responders. Furthermore, the attenuation of pain severity was significantly correlated with the global alpha oscillation activity (r = 0.60, P = 0.002). Likely, positive and significant correlation was found between the pain relief and gamma activity (r = 0.58, P = 0.003). Conclusions Distinct pattern of neural oscillation is associated with the analgesic effect of spinal cord stimulation in pain management, enhancement of cortical alpha and gamma oscillation may be a predictor of analgesia responders.

A quantum theory of stimulated Raman scattering is established that takes into account the time-dependence of pump laser pulse, two-photon detuning and atomic populations, allowing the general transient and spatial propagation study of Raman memory. Heisenberg–Langevin and Maxwell equations for the collective atomic operators are solved analytically under adiabatic condition, where the electric field envelopes and the collective atomic operators vary slowly. The analytical solution of Stokes/anti-Stokes signal electric field operator is obtained. The spatial–temporal intensity of quantum signal field is studied numerically and analytically for different two-photon detunings and initial populations.

Abstract Purpose In soft tissue reconstructive surgery, perforator localization and flap harvesting have always been critical challenges, but augmented reality (AR) has become a dominant technology to help map perforators. Methods The lateral circumflex femoral artery (LCFA) and its perforators were reconstructed by CTA in consecutive patients (N = 14). Then, the anterolateral thigh perforators and the points from which the perforators emerged from the deep fascia were marked and projected onto the skin surface. As the virtual images were projected onto patients according to bony markers, the courses of the LCFA and its perforators were depicted on the skin surface for intraoperative guidance. Finally, the locations of the emergence points were verified by intraoperative findings and compared to those determined by handheld Doppler ultrasound. Results The sources, locations, and numbers of perforators were determined by CTA. The perforators and their emergence points were accurately mapped on the skin surface by a portable projector to harvest the anterolateral thigh perforator flap. During the operation, the accuracy of the CTA & AR method was 90.2% (37/41), and the sensitivity reached 97.4% (37/38), which were much higher than the corresponding values of Doppler ultrasound. Additionally, the differences between the AR-marked points and the intraoperative findings were much smaller than those seen with Doppler ultrasound (P

Abstract Background How to treat the posterior cruciate ligament (PCL) tibial insertion small and comminuted avulsion fracture is still challenging. Our study evaluated the clinical and radiological outcomes after ORIF of PCL tibial insertion avulsion fractures through the inverted L-shaped postero-medial approach using a homemade pin-hook. Methods Between January 2009 and December 2020, twenty-four patients with isolated PCL tibial insertion bony avulsion were enrolled. There were 16 males and 8 females. The age range was 18-48 (32.5 ± 9.3) years. The time from injury to surgery was 1-10 (4.4 ± 2.8) days. There were 11 cases in the left knee and 13 cases in the right knee. The patients received anticoagulant therapy to prevent thrombosis. Preoperative standard X-ray, computerized tomography (CT) and magnetic resonance imaging (MRI) were performed. According to the Meyers-McKeever classification, there were 8 cases of type II and 16 cases of type III. Results The operation time was 60-120 (89.6 ± 19.8) min. Postoperative follow-up ranged from 3 to 18 months. The average follow-up was 11.4 ± 4.3 months. In all patients, one or two homemade pin-hooks were used to fix different sizes of fracture segments. X-ray or CT scans taken after surgery revealed fracture union. The fractures healed in 9-16 (11.8 ± 1.7) weeks. At the last follow-up, the patients were able to fully straighten. The ROM (132.6° ± 3.9°), the Tegner-Lysholm score (96.2 ± 2.3) and the IKDC scores (95.5 ± 1.6) were all significantly improved compared with the preoperative values (77.5° ± 13.1°, 46.8 ± 8.9, 36.2 ± 7.9). The posterior drawer test was negative. The gastrocnemius muscle strength did not diminish. No internal fixation migration was observed during the follow-up. No neurovascular bundle- or hardware-related complications were reported. Conclusions The inverted L-shaped postero-medial approach with homemade pin-hook fixation for the treatment of PCL avulsion fractures produces acceptable clinical and radiological results. Moreover, the homemade pin-hook made of K-wires is affordable and reduces patient costs. It is a practical application and worth recommending, especially for community hospitals.

Objectives: Studies reported associations of inflammatory markers with the severity of COVID-19, but conclusions were inconsistent. We aimed to provide an overview of the association of inflammatory markers with the severity of COVID-19. Methods: We searched PubMed, Embase, Cochrane Library, Wanfang and China National Knowledge Infrastructure (CNKI) database until March 20, 2020. Weighted mean difference (WMD) and 95% confidence intervals (CIs) were pooled using random or fixed-effects models. Results: A total of 16 studies comprising 3962 patients with COVID-19 were included in our analysis. Random-effect results demonstrated that patients with COVID-19 in the nonsevere group had lower levels for CRP (WMD = −41.78 mg/l, 95% CI = [−52.43, −31.13], P < 0.001), PCT (WMD = −0.13 ng/ml, 95% CI = [−0.20, −0.05], P < 0.001), IL-6 (WMD = −21.32 ng/l, 95% CI = [−28.34, −14.31], P < 0.001), ESR (WMD = −8 mm/h, 95% CI = [−14, −2], P = 0.005), SAA (WMD = −43.35 μg/ml, 95% CI = [−80.85, −5.85], P = 0.020) and serum ferritin (WMD = −398.80 mg/l, 95% CI = [−625.89, −171.71], P < 0.001), compared with those in the severe group. Moreover, survivors had a lower level of IL-6 than non-survivors (WMD = −4.80 ng/ml, 95% CI = [−5.87, −3.73], P < 0.001). These results were consistent through sensitivity analysis and publication bias assessment. Conclusions: The meta-analysis highlights the association of inflammatory markers with the severity of COVID-19. Measurement of inflammatory markers might assist clinicians to monitor and evaluate the severity and prognosis of COVID-19.

BackgroundMinimally invasive techniques, such as percutaneous low-power laser discectomy (PLLD) and low-temperature plasma radiofrequency ablation (coblation) can be applied to treat degenerative cervical radiculopathy. However, less evidence supports the superiority of distinct minimally-invasive therapy. Our study aimed to evaluate the clinical and radiological characteristics of the PLLD and coblation for cervical radiculopathy.MethodsThis was a prospective, multicenter, cohort study (ChiCTR-ONC-17010356). The modified Macnab criteria was performed to assess the clinical improvement pre- and post-surgery. To evaluate the radiological effect, the Pfirrmann grading system and disk herniation index were applied with MRI.ResultsIn this study, 28 patients were enrolled in the coblation group and 30 patients in the PLLD group. The mean good-excellent rate at 3-month follow-up was 82.1% for PLLD group, and 66.7% for coblation group, respectively (p = 0.179). The PLLD group achieved higher good-excellent rate 6 and 12 months after discharge (92.9 vs. 70.0%, p = 0.026). Radiological data revealed that PLLD but not coblation treatment achieved significant reduction of disk herniation index (p < 0.0001). Coblation treatment did not change the Pfirrmann grades of cervical radiculopathy patients (n = 18), and 7 out of 17 (41.2%) patients achieved improvement after PLLD therapy. None obvious adverse event was observed in this study.ConclusionBoth PLLD and coblation are effective and safe option for patients with cervical radiculopathy. Better long-term clinical outcomes may be potentially associated with the improvement of disk degeneration after PLLD treatment.

Oxygen evolution reaction is a key process in hydrogen production from water splitting. The development of non-noble metal electrode materials with high efficiency and low cost has become the key factor for large-scale hydrogen production. Binary NiCo-layered double hydroxide (LDH) has been used as a non-noble metal electrocatalyst for OER, but its overpotential is still large. The microstructure of the catalyst is tuned by doping Mo ions into the NiCo-LDH/NF nanowires to form ternary NiCoMo-LDH/NF nanosheet catalysts for the purpose of enhancing the active sites and reducing the initial overpotential. Only 1.5 V (vs. reversible hydrogen electrode (RHE), ≈270 mV overpotential) is required to achieve a catalytic current density of 10 mA cm−2 and a small Tafel slope of 81.46 mV dec−1 in 1 M KOH solution, which manifests the best performance of NiCo-based catalysts reported up to now. Electrochemical analysis and micro-morphology show that the high catalytic activity of NiCoMo-LDH/NF is attributable to the change of the microstructure. The interconnected nanosheet arrays have the obvious advantages of electrolyte diffusion and ion migration. Thus, the active sites of catalysts are significantly increased, which facilitates the adsorption and desorption of intermediates. We conclude that NiCoMo-LDH/NF is a promising electrode material for its low cost and excellent electrocatalytic properties.

BackgroundLumbosacral radicular pain (LSRP) can be caused by disc herniation, spinal stenosis, and failed back surgery syndrome. The clinical effect of pulsed-radiofrequency (PRF) combined with transforaminal epidural steroid injection (TESI) for radiating pain in different population remains unclear.MethodsWe retrospectively reviewed the medical recordings of patients with LSRP caused by different etiologies, who underwent PRF and TESI treatment. The primary clinical outcome was assessed by a 10-point Visual Analog Scale (VAS) pre- and post-treatment.ResultsA total of 34 LSRP patients were identified and classified into 3 subgroups (disc herniation, spinal stenosis, and failed back surgery syndrome). The overall immediate pain reduction was 4.4 ± 1.1 after procedure. After a median follow-up of 9.5 months, the VAS decreased from 6.5 ± 1.0 to 2.4 ± 1.9 at the last follow-up.ConclusionPRF combined with TESI is an effective approach to treat persistent LSRP in distinct population.

Autologous chondrocyte implantation (ACI) is a procedure used to treat articular cartilage injuries and prevent the onset of post-traumatic osteoarthritis. In vitro expansion of chondrocytes, a necessary step in ACI, results in the generation of senescent cells that adversely affect the quality and quantity of newly formed cartilage. Recently, a senolytic peptide, fork head box O transcription factor 4-D-Retro-Inverso (FOXO4-DRI), was reported to selectively kill the senescent fibroblasts. In this study, we hypothesized that FOXO4-DRI treatment could remove the senescent cells in the expanded chondrocytes, thus enhancing their potential in generating high-quality cartilage. To simulate the in vitro expansion for ACI, chondrocytes isolated from healthy donors were expanded to population doubling level (PDL) 9, representing chondrocytes ready for implantation. Cells at PDL3 were also used to serve as the minimally expanded control. Results showed that the treatment of FOXO4-DRI removed more than half of the cells in PDL9 but did not significantly affect the cell number of PDL3 chondrocytes. Compared to the untreated control, the senescence level in FOXO4-DRI treated PDL9 chondrocytes was significantly reduced. Based on the result from standard pellet culture, FOXO4-DRI pre-treatment did not enhance the chondrogenic potential of PDL9 chondrocytes. However, the cartilage tissue generated from FOXO4-DRI pretreated PDL9 cells displayed lower expression of senescence-relevant secretory factors than that from the untreated control group. Taken together, FOXO4-DRI is able to remove the senescent cells in PDL9 chondrocytes, but its utility in promoting cartilage formation from the in vitro expanded chondrocytes needs further investigation.

Background. Knee osteoarthritis (KOA) is a common degenerative disease associated with joint dysfunction and pain. Ultrasound-guided radiofrequency (RF) may be a promising therapy in the treatment of chronic pain for KOA patients. Objective. To evaluate the efficacy and safety of ultrasound-guided RF treatment for chronic pain in patients with KOA. Design. A systematic review was conducted, and a meta-analysis was carried out when possible. Setting. We examined the studies evaluating the clinical efficiency of ultrasound-guided RF on chronic pain in KOA population. Method. A systematic review for the efficacy and safety of ultrasound-guided RF treatment for pain management of KOA patients was carried out in PubMed, EMBASE, Cochrane Library, Web of Science, Wanfang Data, and China National Knowledge Infrastructure (CNKI) from the date of inception to February 2020, and a meta-analysis was conducted. The primary outcomes of pain intensity (visual analogue scale or numerical rating scale) and knee function [the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC)] were evaluated from baseline to various follow-up times by random-effects model. Heterogeneity was assessed by I2 statistic and the potential sources of heterogeneity by subgroup and metaregression analyses, respectively. Results. Eight publications with 256 patients were included in the meta-analysis. RF could relieve pain with −4.196 of pooled mean difference and improve knee function by decreasing 23.155 points in WOMAC. Three patients had ecchymosis, two with hypoesthesia and one with numbness after the procedure, and improved within 6 months. Furthermore, study design and treatment target were the sources of heterogeneity by subgroup and metaregression analyses, accounting for 37% and 74% of variances, respectively. Target of genicular nerve achieved better pain relief than intra-articular or sciatic nerve. Sensitivity analysis showed that removal of any single study was unlikely to overturn the findings. Limitations. There were some limitations in the study. Firstly, the small number of relevant studies limited the confidence level of the meta-analysis. Also, the significant heterogeneity may not be explained due to the limited data. Secondly, the direct comparison of two different guidance methods (ultrasound vs. fluoroscopy) for RF therapy is lacking. In addition, the outcomes were blindly assessed in the meta-analysis from all studies according to evaluation of bias, which could affect the reality of the data. Finally, most of the studies only provided short follow-up times, so we could not analyze the long-term effectiveness of ultrasound-guided RF in the treatment of patients with KOA. Conclusions. Ultrasonography is an effective, safe, nonradiative, and easily applicable guidance method for RF in pain relief and functional improvement in KOA patients.

(1) Background: Chronic kidney disease (CKD) increases the susceptibility to the presence of herpes zoster (HZ). Little is known about the risk factors of HZ in CKD patients; (2) Methods: This is a case-control study. CKD patients diagnosed with HZ between January 2015 and June 2021 in a tertiary hospital were identified. One age- and gender- matched control was paired for each case, matched to the date of initial HZ diagnosis. Conditional multiple logistic regression was used to evaluate the risk factors associated with the presence of HZ; (3) Results: Forty-seven HZ patients and controls were identified. In general, about 73.40% (69 out of 94) patients were classified at IV to V stages of CKD. Immunosuppressive agents (p = 0.0012) and dialysis therapy (p = 0.021) were reported more frequently in the HZ cohort. Compared with the control group, the total white cell count and lymphocyte count were significantly lower in the HZ group (p value of 0.032 and 0.003, respectively). The conditional logistics regression model revealed that previous immunosuppressants administration (odds ratio: 10.861, 95% CI: 2.092~56.392, p = 0.005) and dialysis therapy (odds ratio: 3.293, 95% CI: 1.047~10.355, p = 0.041) were independent risk factors of HZ in the CKD population; (4) Conclusions: Dialysis and immunosuppressants therapy were associated with greater risk of HZ disease in CKD patients. Further guideline may highlight the necessity of zoster vaccine for patients with CKD, who undertake associated treatment.

Despite the importance of WRKY genes in plant physiological processes, little is known about their roles in Panax ginseng C.A. Meyer. Forty-eight unigenes on this species were previously reported as WRKY transcripts using the next-generation sequencing (NGS) technology. Subsequently, one gene that encodes PgWRKY1 protein belonging to subgroup II-d was cloned and functionally characterized. In this study, eight WRKY genes from the NGS-based transcriptome sequencing dataset designated as PgWRKY2-9 have been cloned and characterized. The genes encoding WRKY proteins were assigned to WRKY Group II (one subgroup II-c, four subgroup II-d, and three subgroup II-e) based on phylogenetic analysis. The cDNAs of the cloned PgWRKYs encode putative proteins ranging from 194 to 358 amino acid residues, each of which includes one WRKYGQK sequence motif and one C2H2-type zinc-finger motif. Quantitative real-time PCR (qRT-PCR) analysis demonstrated that the eight analyzed PgWRKY genes were expressed at different levels in various organs including leaves, roots, adventitious roots, stems, and seeds. Importantly, the transcription responses of these PgWRKYs to methyl jasmonate (MeJA) showed that PgWRKY2, PgWRKY3, PgWRKY4, PgWRKY5, PgWRKY6, and PgWRKY7 were downregulated by MeJA treatment, while PgWRKY8 and PgWRKY9 were upregulated to varying degrees. Moreover, the PgWRKY genes increased or decreased by salicylic acid (SA), abscisic acid (ABA), and NaCl treatments. The results suggest that the PgWRKYs may be multiple stress–inducible genes responding to both salt and hormones.

Tissue-specific loss-of-function (LOF) analysis is essential for characterizing gene function. Here, we present a simple, yet highly efficient, clustered regularly interspaced short palindromic repeats (CRISPR)-mediated tissue-restricted mutagenesis (CRISPRTRiM) method for ablating gene function in Drosophila. This binary system consists of a tissue-specific Cas9 and a ubiquitously expressed multi-guide RNA (gRNA) transgene. We describe convenient toolkits for making enhancer-driven Cas9 lines and multigRNAs that are optimized for mutagenizing somatic cells. We demonstrate that insertions or deletions in coding sequences more reliably cause somatic mutations than DNA excisions induced by two gRNAs. We further show that enhancer-driven Cas9 is less cytotoxic yet results in more complete LOF than Gal4-driven Cas9 in larval sensory neurons. Finally, CRISPR-TRiM efficiently unmasks redundant soluble N-ethylmaleimide-sensitive factor attachment protein receptor gene functions in neurons and epidermal cells. Importantly, Cas9 transgenes expressed at different times in the neuronal lineage reveal the extent to which gene products persist in cells after tissue-specific gene knockout. These CRISPR tools can be applied to analyze tissue-specific gene function in many biological processes. [ABSTRACT FROM AUTHOR]