Microfluidic impedance cytometry with flat-end cylindrical electrodes for accurate and fast analysis of marine microalgae.

Marine microalgae play an increasingly significant role in addressing the issues of environmental monitoring and disease treatment, making the analysis of marine microalgae at the single-cell level an essential technique. For this, we put forward accurate and fast microfluidic impedance cytometry to analyze microalgal cells by assembling two cylindrical electrodes and microchannels to form a three-dimensional detection zone. Firstly, we established a mathematical model of microalgal cell detection based on Maxwell's mixture theory and numerically investigated the effects of the electrode gap, microalgal positions, and ion concentrations of the solution on detection to optimize detection conditions. Secondly, 80 µm stainless steel wires were used to construct flat-ended cylindrical electrodes and were then inserted into two collinear channels fabricated using standard photolithography techniques to form a spatially uniform electric field to promote the detection throughput and sensitivity. Thirdly, based on the validation of this method, we measured the impedance of living Euglena and Haematococcus pluvialis to study parametric influences, including ion concentration, cell density and electrode gap. The throughput of this method was also investigated, which reached 1800 cells per s in the detection of Haematococcus pluvialis. Fourthly, we analyzed live and dead Euglena to prove the ability of this method to detect the physiological status of cells and obtained impedances of 124.3 Ω and 31.0 Ω with proportions of 15.9% and 84.1%, respectively. Finally, this method was engineered for the analysis of marine microalgae, measuring living Euglena with an impedance of 159.61 Ω accounting for 3.9%, dead Euglena with an impedance of 36.43 Ω accounting for 10.1% and Oocystis sp. with an impedance of 55.00 Ω accounting for about 81.0%. This method could provide a reliable tool to analyze marine microalgae for monitoring the marine environment and treatment of diseases owing to its outstanding advantages of low cost, high throughput and high corrosion resistance.

Effect of pyrolysis temperature and molecular weight on characterization of biochar derived dissolved organic matter from invasive plant and binding behavior with the selected pharmaceuticals.

A comprehensive understanding of the characteristics of biochar released-dissolved organic matter (BDOM) derived from an invasive plant and its impact on the binding behavior of pharmaceuticals is essential for the application of biochar, yet has received less attention. In this study, the binding behavior of BDOM pyrolyzed at 300-700 °C with sulfathiazole, acetaminophen, chloramphenicol (CAP), and carbamazepine (CMZ) was investigated based on a multi-analytical approach. Generally, the pyrolysis temperature exhibited a more significant impact on the spectral properties of BDOM and pharmaceutical binding behavior than those of the molecular weight. With increased pyrolysis temperature, the dissolved organic carbon decreased while the proportion of the protein-like substance increased. The highest binding capacity towards the drugs was observed for the BDOM pyrolyzed at 500 °C with the molecular weight larger than 0.3 kDa. Moreover, the protein-like substance exhibited higher susceptive and released preferentially during the dialysis process and also showed more sensitivity and bound precedingly with the pharmaceuticals. The active binding points were the aliphatic C-OH, amide II N-H, carboxyl CO, and phenolic-OH on the tryptophan-like substance. Furthermore, the binding affinity of the BDOM pyrolyzed at 500 °C was relatively high with the stability constant (logKM) of 4.51 ± 0.52.

Interspecies/Intergroup Complementation of Orthotospovirus Replication and Movement through Reverse Genetics Systems.

Orthotospoviruses, the plant-infecting bunyaviruses, cause serious diseases in agronomic crops and pose major threats to global food security. The family of Tospoviridae contains more than 30 members that are classified into two geographic groups, American-type and Euro/Asian-type orthotospovirus. However, the genetic interaction between different species and the possibility, during mixed infections, for transcomplementation of gene functions by orthotospoviruses from different geographic groups remains underexplored. In this study, minireplicon-based reverse genetics (RG) systems have been established for Impatiens necrotic spot virus (INSV) (an American-type orthotospovirus) and for Calla lily chlorotic spot virus and Tomato zonate spot virus (CCSV and TZSV) (two representative Euro/Asian orthotospoviruses). Together with the earlier established RG system for Tomato spotted wilt virus (TSWV), a type species of the Orthotospovirus American-clade, viral replicase/movement proteins were exchanged and analyzed on interspecies transcomplementation. Whereas the homologous RNA-dependent RNA polymerase (RdRp) and nucleocapsid (N) protein supported the replication of orthotospoviruses from both geographic groups, heterologous combinations of RdRp from one group and N from the other group were unable to support the replication of viruses from both groups. Furthermore, the NSm movement protein (MP), from both geographic groups of orthotospoviruses, was able to transcomplement heterologous orthotospoviruses or a positive-strand Cucumber mosaic virus (CMV) in their movement, albeit with varying efficiency. MP from Rice stripe tenuivirus (RSV), a plant-infecting bunyavirus that is distinct from orthotospoviruses, or MP from CMV also moves orthotospoviruses. Our findings gain insights into the genetic interaction/reassortant potentials for the segmented plant orthotospoviruses. IMPORTANCE Orthotospoviruses are agriculturally important negative-strand RNA viruses and cause severe yield-losses on many crops worldwide. Whereas the emergence of new animal-infecting bunyaviruses is frequently associated with genetic reassortants, this issue remains underexposed with the plant-infecting orthotospovirus. With the development of reverse genetics systems for orthotospoviruses from different geographic regions, the interspecies/intergroup replication/movement complementation between American- and Euro/Asian-type orthotospoviruses were investigated. Genomic RNAs from American orthotospoviruses can be replicated by the RdRp and N from those of Euro/Asia-group orthotospoviruses, and vice versa. However, their genomic RNAs cannot be replicated by a heterologous combination of RdRp from one geographic group and N from another geographic group. Cell-to-cell movement of viral entity is supported by NSm from both geographic groups, with highest efficiency by NSm from viruses belonging to the same group. Our findings provide important insights into the genetic interaction and exchange ability of viral gene functions between different species of orthotospovirus.

Neoadjuvant chemoradiotherapy with camrelizumab in patients with locally advanced esophageal squamous cell carcinoma.

Background: Neoadjuvant anti-programmed death receptor-1 (PD-1) blockade has been reported to improve the prognosis of locally advanced esophageal squamous cell carcinoma (ESCC). This study was aimed to evaluate the efficacy and safety of neoadjuvant camrelizumab plus chemoradiotherapy in locally advanced ESCC. Methods: We retrospectively enrolled ESCC patients who received camrelizumab plus chemoradiotherapy as neoadjuvant therapy before surgery from May 2019 to September 2021. Results: A total of 38 eligible patients were enrolled. The neoadjuvant treatment was well tolerated with no serious treatment-related adverse events. 36 (94.7%) patients achieved a R0 resection without hospital mortality or any other serious intraoperative complications. The objective response rate (ORR) was 63.2% and the disease control rate (DCR) was 100.0%. The major pathological response (MPR) was 50.0% and the complete pathological response (pCR) was 39.5%. With a median follow-up of 18.5 months, 6 (15.8%) patients had died. The overall survival (OS) and disease-free survival (DFS) at 12 months were 87.6% and 78.7%, respectively. Subgroup analysis demonstrated that patients who got MPR or pCR achieved improved survival, while PD-L1 expression did not reach statistically difference in predicting survival. Conclusions: Neoadjuvant camrelizumab plus chemoradiotherapy is safe and efficacious in treating patients with locally advanced ESCC.

[Advances in biodegradation of macrolide antibiotics].

Macrolide antibiotics are a class of broad-spectrum antibiotics with the macrolide as core nucleus. Recently, antibiotic pollution has become an important environmental problem due to the irregular production and abuse of macrolide antibiotics. Microbial degradation is one of the most effective methods to deal with antibiotic pollution. This review summarizes the current status of environmental pollution caused by macrolide antibiotics, the degradation strains, the degradation enzymes, the degradation pathways and the microbial processes for degrading macrolide antibiotics. Moreover, the critical challenges on the biodegradation of macrolide antibiotics were also discussed.

Development of a Mini-Replicon-Based Reverse-Genetics System for Rice Stripe Tenuivirus.

Negative-stranded RNA (NSR) viruses include both animal- and plant-infecting viruses that often cause serious diseases in humans and livestock and in agronomic crops. Rice stripe tenuivirus (RSV), a plant NSR virus with four negative-stranded/ambisense RNA segments, is one of the most destructive rice pathogens in many Asian countries. Due to the lack of a reliable reverse-genetics technology, molecular studies of RSV gene functions and its interaction with host plants are severely hampered. To overcome this obstacle, we developed a mini-replicon-based reverse-genetics system for RSV gene functional analysis in Nicotiana benthamiana. We first developed a mini-replicon system expressing an RSV genomic RNA3 enhanced green fluorescent protein (eGFP) reporter [MR3(-)eGFP], a nucleocapsid (NP), and a codon usage-optimized RNA-dependent RNA polymerase (RdRpopt). Using this mini-replicon system, we determined that RSV NP and RdRpopt are indispensable for the eGFP expression from MR3(-)eGFP. The expression of eGFP from MR3(-)eGFP can be significantly enhanced in the presence of four viral suppressors of RNA silencing (VSRs), NSs, and P19-HcPro-γb. In addition, NSvc4, the movement protein of RSV, facilitated eGFP trafficking between cells. We also developed an antigenomic RNA3-based replicon in N. benthamiana. However, we found that the RSV NS3 coding sequence acts as a cis element to regulate viral RNA expression. Finally, we made mini-replicons representing all four RSV genomic RNAs. This is the first mini-replicon-based reverse-genetics system for monocot-infecting tenuivirus. We believe that the mini-replicon system described here will allow studies of the RSV replication, transcription, cell-to-cell movement, and host machinery underpinning RSV infection in plants. IMPORTANCE Plant-infecting segmented negative-stranded RNA (NSR) viruses are grouped into three genera: Orthotospovirus, Tenuivirus, and Emaravirus. Reverse-genetics systems have been established for members of the genera Orthotospovirus and Emaravirus. However, there is still no reverse-genetics system available for Tenuivirus. Rice stripe virus (RSV) is a monocot-infecting tenuivirus with four negative-stranded/ambisense RNA segments. It is one of the most destructive rice pathogens and causes significant damage to the rice industry in Asian countries. Due to the lack of a reliable reverse-genetics system, molecular characterizations of RSV gene functions and the host machinery underpinning RSV infection in plants are extremely difficult. To overcome this obstacle, we developed a mini-replicon-based reverse-genetics system for RSV in Nicotiana benthamiana. This is the first mini-replicon-based reverse-genetics system for tenuivirus. We consider that this system will provide researchers a new working platform to elucidate the molecular mechanisms dictating segmented tenuivirus infections in plants.

Sensitive time-resolved fluoroimmunoassay for quantitative determination of clothianidin in agricultural samples.

Europium (Eu(3+))-labeled antibody was used as a fluorescent label to develop a highly sensitive time-resolved fluoroimmunoassay (TRFIA) for determination of clothianidin residues in agricultural samples. Toward this goal, the Eu(3+)-labeled polyclonal antibody and goat anti-rabbit antibody were prepared for developing and evaluating direct competitive TRFIA (dc-TRFIA) and indirect competitive TRFIA (ic-TRFIA). Under optimal conditions, the half-maximal inhibition concentration (IC50) and the limit of detection (LOD, IC10) of clothianidin were 9.20 and 0.0909 µg/L for the dc-TRFIA and 2.07 and 0.0220 µg/L for the ic-TRFIA, respectively. The ic-TRFIA has no obvious cross-reactivity with the analogues of clothianidin except for dinotefuran. The average recoveries of clothianidin from spiked water, soil, cabbage, and rice samples were estimated to range from 74.1 to 115.9 %, with relative standard deviations of 3.3 to 11.7 %. The results of TRFIA for the blind samples were largely consistent with gas chromatography (R (2) = 0.9902). The optimized ic-TRFIA might become a sensitive and satisfactory analytical method for the quantitative monitoring of clothianidin residues in agricultural samples.