Scaffold RNA engineering in type V CRISPR-Cas systems: a potent way to enhance gene expression in the yeast Saccharomyces cerevisiae.

New, orthogonal transcription factors in eukaryotic cells have been realized by engineering nuclease-deficient CRISPR-associated proteins and/or their guide RNAs. In this work, we present a new kind of orthogonal transcriptional activators, in Saccharomyces cerevisiae, made by turning type V CRISPR RNA into a scaffold RNA (ScRNA) able to recruit a variable number of VP64 activation domains. The activator arises from the complex between the synthetic ScRNA and DNase-deficient type V Cas proteins: dCas12e and denAsCas12a. The transcription activation achieved via the newly engineered dCas:ScRNA system is up to 4.7-fold higher than that obtained with the direct fusion of VP64 to Cas proteins. The new transcription factors have been proven to be functional in circuits such as Boolean gates, converters, multiplex-gene and metabolic-pathway activation. Our results extend the CRISPR-Cas-based technology with a new effective tool that only demands RNA engineering and improves the current design of transcription factors based on type V Cas proteins.

CRISPR-associated type V proteins as a tool for controlling mRNA stability in S. cerevisiae synthetic gene circuits.

Type V-A CRISPR-(d)Cas system has been used in multiplex genome editing and transcription regulation in both eukaryotes and prokaryotes. However, mRNA degradation through the endonuclease activity of Cas12a has never been studied. In this work, we present an efficient and powerful tool to induce mRNA degradation in the yeast Saccharomyces cerevisiae via the catalytic activity of (d)Cas12a on pre-crRNA structure. Our results point out that dFnCas12a, (d)LbCas12a, denAsCas12a and two variants (which carry either NLSs or NESs) perform significant mRNA degradation upon insertion of pre-crRNA fragments into the 5'- or 3' UTR of the target mRNA. The tool worked well with two more Cas12 proteins-(d)MbCas12a and Casϕ2-whereas failed by using type VI LwaCas13a, which further highlights the great potential of type V-A Cas proteins in yeast. We applied our tool to the construction of Boolean NOT, NAND, and IMPLY gates, whose logic operations are fully based on the control of the degradation of the mRNA encoding for a reporter protein. Compared to other methods for the regulation of mRNA stability in yeast synthetic gene circuits (such as RNAi and riboswitches/ribozymes), our system is far easier to engineer and ensure very high performance.

Detection and diagnosis of automated breast ultrasound in patients with BI-RADS category 4 microcalcifications: a retrospective study.

BACKGROUND: Automated Breast Ultrasound (AB US) has shown good application value and prospects in breast disease screening and diagnosis. The aim of the study was to explore the ability of AB US to detect and diagnose mammographically Breast Imaging Reporting and Data System (BI-RADS) category 4 microcalcifications. METHODS: 575 pathologically confirmed mammographically BI-RADS category 4 microcalcifications from January 2017 to June 2021 were included. All patients also completed AB US examinations. Based on the final pathological results, analyzed and summarized the AB US image features, and compared the evaluation results with mammography, to explore the detection and diagnostic ability of AB US for these suspicious microcalcifications. RESULTS: 250 were finally confirmed as malignant and 325 were benign. Mammographic findings including microcalcifications morphology (61/80 with amorphous, coarse heterogeneous and fine pleomorphic, 13/14 with fine-linear or branching), calcification distribution (189/346 with grouped, 40/67 with linear and segmental), associated features (70/96 with asymmetric shadow), higher BI-RADS category with 4B (88/120) and 4 C (73/38) showed higher incidence in malignant lesions, and were the independent factors associated with malignant microcalcifications. 477 (477/575, 83.0%) microcalcifications were detected by AB US, including 223 malignant and 254 benign, with a significantly higher detection rate for malignant lesions (x2 = 12.20, P < 0.001). Logistic regression analysis showed microcalcifications with architectural distortion (odds ratio [OR] = 0.30, P = 0.014), with amorphous, coarse heterogeneous and fine pleomorphic morphology (OR = 3.15, P = 0.037), grouped (OR = 1.90, P = 0.017), liner and segmental distribution (OR = 8.93, P = 0.004) were the independent factors which could affect the detectability of AB US for microcalcifications. In AB US, malignant calcification was more frequent in a mass (104/154) or intraductal (20/32), and with ductal changes (30/41) or architectural distortion (58/68), especially with the both (12/12). BI-RADS category results also showed that AB US had higher sensitivity to malignant calcification than mammography (64.8% vs. 46.8%). CONCLUSIONS: AB US has good detectability for mammographically BI-RADS category 4 microcalcifications, especially for malignant lesions. Malignant calcification is more common in a mass and intraductal in AB US, and tend to associated with architectural distortion or duct changes. Also, AB US has higher sensitivity than mammography to malignant microcalcification, which is expected to become an effective supplementary examination method for breast microcalcifications, especially in dense breasts.

Therapeutic potential of coumestan Pks13 inhibitors for tuberculosis.

Polyketide synthase 13 (Pks13) is an important enzyme found in Mycobacterium tuberculosis (M. tuberculosis) that condenses two fatty acyl chains to produce α-alkyl ß-ketoesters, which in turn serve as the precursors for the synthesis of mycolic acids that are essential building blocks for maintaining the cell wall integrity of M. tuberculosis Coumestan derivatives have recently been identified in our group as a new chemotype that exert their antitubercular effects via targeting of Pks13. These compounds were active on both drug-susceptible and drug-resistant strains of M. tuberculosis as well as showing low cytotoxicity to healthy cells and a promising selectivity profile. No cross-resistance was found between the coumestan derivatives and first-line TB drugs. Here we report that treatment of M. tuberculosis bacilli with 15 times the MIC of compound 1, an optimized lead coumestan compound, resulted in a colony forming unit (CFU) reduction from 6.0 log10 units to below the limit of detection (1.0 log10 units) per mL culture, demonstrating a bactericidal mechanism of action. Single dose (10 mg/kg) pharmacokinetic studies revealed favorable parameters with a relative bioavailability of 19.4%. In a mouse infection and chemotherapy model, treatment with 1 showed dose-dependent mono-therapeutic activity, whereas treatment with 1 in combination with rifampin showed clear synergistic effects. Together these data suggest that coumestan derivatives are promising agents for further TB drug development.

Value of multimodal imaging in the diagnosis of breast sclerosing adenosis associated with malignant lesions.

AIM: To explore the diagnostic value of multimodal imaging techniques, including automatic breast volume scanner (ABVS), mammography (MG), and magnetic resonance (MRI) in breast sclerosing adenosis (SA) associated with malignant lesions. METHODS: From January 2018 to October 2020, 76 patients (88 lesions) with pathologically confirmed as SA associated with malignant or benign lesions were retrospective analyzed. All patients completed ABVS examination, 58 patients (67 lesions) with MG and 50 patients (62 lesions) with MRI were also completed before biopsy or surgical excision, of which, six patients (eight lesions) diagnosed as Breast Imaging Reporting and Data System (BI-RADS) category 3 by all imaging examinations underwent surgical excision without biopsy, other 70 patients (80 lesions) with BI-RADS category 4 or above by any imaging examination completed biopsy, including 65 patients (75 lesions) were further surgical excised and the other five patients (five lesions) were just followed up. All lesions were retrospectively described and classified, and were divided into benign group and malignant group according to their pathological results. Image features of different examination methods between the two groups were compared and analyzed. A ROC curve was established using the sensitivity of BI-RADS categories to predict malignant lesions in different imaging techniques as the ordinate and 1-specificity as the abscissa. RESULTS: 88 lesions including 26 purely SA and 45 SA associated with benign lesions were classified as benign group, and the remaining 17 SA associated with malignant lesions were classified as malignant group. On ABVS, 40 mass lesions, their heterogeneous echo, not circumscribed margin and coronal convergence signs were statistically significant for malignant lesions (p < .05), but the remain 48 nonmass lesions lack specific sonographic features. On MG, 12 showed negative results, 55 showed with microcalcification, mass, structural distortion, and asymmetric density shadow, of which 11 lesions had the above two signs at the same time, but only microcalcification had statistical difference between the two groups. 35 mass enhanced lesions and 27 nonmass enhanced lesions on MRI, but there were no significant difference between their pathological results. Time signal intensity curves showed no differences, but ADC value <1.10 × 10-3  mm2 /s is more significant in malignant lesions (p < .05). The area under the ROC curve (AUC) of BI-RADS classification of ABVS, MG, and MRI in the diagnosis of malignant lesions were 0.611, 0.474, and 0.751, respectively, and the AUC of the combined diagnosis of the three was 0.761. CONCLUSION: Mass lesions with heterogeneous echo, not circumscribed margin and coronal convergence sign on ABVS, microcalcification on MG and the ADC value <1.10 × 10-3  mm2 /s on MRI are significant signs for SA associated with malignant lesions. The combined diagnosis of the three methods was the highest, and the following were MRI, ABVS, and MG. Therefore, be cognizant of significant characteristics in SA associated with malignancy showed in different imaging examinations can improve the preoperative evaluation of SA and better provide basis for subsequent clinical decision-making.

Efficiency and impact factors of anatomical intelligence for breast and hand-held ultrasound in lesion detection.

PURPOSE: To investigate the efficiency and impact factors of anatomical intelligence for breast (AI-Breast) and hand-held ultrasound (HHUS) in lesion detection. METHODS: A total of 172 outpatient women were randomly selected, underwent AI-Breast ultrasound (Group AI) once and HHUS twice. HHUS was performed by breast imaging radiologists (Group A) and general radiologists (Group B). For the AI-Breast examination, a trained technician performed the whole-breast scan and data acquisition, while other general radiologists performed image interpretation. The examination time and lesion detection rate were recorded. The impact factors for breast lesion detection, including breast cup size, number of lesions, and benign or malignant lesions were analyzed. RESULTS: The detection rates of Group AI, A, and B were 92.8 ± 17.0%, 95.0 ± 13.6%, and 85.0 ± 22.9%, respectively. Comparable lesion detection rates were observed in Group AI and Group A (P > 0.05), but a significantly lower lesion detection rate was observed in Group B compared to the other two (both P < 0.05). Regarding missed diagnosis rates of malignant lesions, comparable performance was observed in Group AI, Group A, and Group B (8% vs. 4% vs. 14%, all P > 0.05). Scan times of Groups AI, A, and B were 262.15 ± 40.4 s, 237.5 ± 110.3 s, 281.2 ± 86.1 s, respectively. The scan time of Group AI was significantly higher than Group A (P < 0.01), but was slightly lower than Group B (P > 0.05). We found a strong linear correlation between scan time and cup size in Group AI (r = 0.745). No impacts of cup size and number of lesions were found on the lesion detection rate in Group AI (P > 0.05). CONCLUSIONS: With the assist of AI-Breast system, the lesion detection rate of AI-Breast ultrasound was comparable to that of a breast imaging radiologist and superior to that of the general radiologist. AI-Breast ultrasound may be used as a potential approach for breast lesions surveillance.

Synthesis and Biological Evaluation of 3-Amino-4,4-Dimethyl Lithocholic Acid Derivatives as Novel, Selective, and Cellularly Active Allosteric SHP1 Activators.

Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP1), a non-receptor member of the protein tyrosine phosphatase (PTP) family, negatively regulates several signaling pathways that are responsible for pathological cell processes in cancers. In this study, we report a series of 3-amino-4,4-dimethyl lithocholic acid derivatives as SHP1 activators. The most potent compounds, 5az-ba, showed low micromolar activating effects (EC50: 1.54-2.10 µM) for SHP1, with 7.63-8.79-fold maximum activation and significant selectivity over the closest homologue Src homology 2 domain-containing protein tyrosine phosphatase 2 (SHP2) (>32-fold). 5az-ba showed potent anti-tumor effects with IC50 values of 1.65-5.51 µM against leukemia and lung cancer cells. A new allosteric mechanism of SHP1 activation, whereby small molecules bind to a central allosteric pocket and stabilize the active conformation of SHP1, was proposed. The activation mechanism was consistent with the structure-activity relationship (SAR) data. This study demonstrates that 3-amino-4,4-dimethyl lithocholic acid derivatives can be selective SHP1 activators with potent cellular efficacy.

Ultrasound grayscale ratio: a reliable parameter for differentiating between papillary thyroid microcarcinoma and micronodular goiter.

BACKGROUND: The present study aimed to quantify and differentiate the echo levels of papillary thyroid microcarcinomas (PTMCs) and micronodular goiters (MNGs) using the ultrasound grayscale ratio (UGSR) and to investigate the repeatability of UGSR. METHODS: The ultrasound (US) data of 241 patients with 265 PTMCs and 141 patients with 168 MNGs confirmed by surgery and pathology were retrospectively analyzed. All patients had received outpatient ultrasonic examination and preoperative ultrasonic positioning. The RADinfo radiograph reading system was used to measure the grayscales of PTMC, MNG, and thyroid tissues at the same gain level, and the UGSR values of the PTMC, MNG, and thyroid tissue were calculated. The patients were divided into outpatient examination, preoperative positioning, and mean value groups, and the receiver operating characteristic (ROC) curves were calculated to obtain the optimal UGSR threshold to distinguish PTMC from MNG. The interclass correlation coefficient (ICC) was used to assess the consistency of UGSR measured in three groups. RESULTS: The UGSR values of the PTMC and MNG were 0.56 ± 0.14 and 0.80 ± 0.19 (t = 5.84, P < 0.001) in the outpatient examination group, 0.55 ± 0.14 and 0.80 ± 0.19 (t = 18.74, P < 0.001) in the preoperative positioning group, and 0.56 ± 0.12 and 0.80 ± 0.18 (t = 16.49, P < 0.001) in the mean value group. The areas under the ROC curves in the three groups were 0.860, 0.856, and 0.875, respectively. When the UGSR values for the outpatient examination, preoperative positioning, and mean value groups were 0.649, 0.646, and 0.657, respectively, each group obtained its largest Youden index. A reliable UGSR value was obtained between the outpatient examination and preoperative positioning groups (ICC = 0.79, P = 0.68). CONCLUSION: UGSR is a simple and repeatable method to distinguish PTMC from MNG, and hence, can be widely applicable.

Design, Synthesis and Biological Evaluation of N-phenylindole Derivatives as Pks13 Inhibitors againstMycobacterium tuberculosis.

Polyketide synthase 13 (Pks13), an essential enzyme for the survival of Mycobacterium tuberculosis (Mtb), is an attractive target for new anti-TB agents. In our previous work, we have identified 2-phenylindole derivatives against Mtb. The crystallography studies demonstrated that the two-position phenol was solvent-exposed in the Pks13-TE crystal structure and a crucial hydrogen bond was lost while introducing bulkier hydrophobic groups at indole N moieties. Thirty-six N-phenylindole derivatives were synthesized and evaluated for antitubercular activity using a structure-guided approach. The structure-activity relationship (SAR) studies resulted in the discovery of the potent Compounds 45 and 58 against Mtb H37Rv, with an MIC value of 0.0625 µg/mL and 0.125 µg/mL, respectively. The thermal stability analysis showed that they bind with high affinity to the Pks13-TE domain. Preliminary ADME evaluation showed that Compound 58 displayed modest human microsomal stability. This report further validates that targeting Pks13 is a valid strategy for the inhibition of Mtb and provides a novel scaffold for developing leading anti-TB compounds.

Enantioselective synthesis of chiral 3-alkyl-3-nitro-4-chromanones via chiral thiourea-catalysed intramolecular Michael-type cyclization.

Herein we report an enantioselective method for the rapid construction of chiral 3-nitro-4-chromanones via a chiral thiourea-catalyzed intramolecular Michael-type cyclization reaction. With this method, a series of 3,3-disubstituted-3-nitro-4-chromanones bearing contiguous C2/C3 stereocenters were obtained with high diastereoselectivities and good to excellent enantioselectivities. In vitro biological evaluations indicated that the chiral amide derivative of the product showed more potent antitumor activities than both the racemic and the corresponding enantiomers, showcasing the high influence of enantioselective methodology development toward medicinal studies.

Imaging manifestations of Caroli disease with autosomal recessive polycystic kidney disease: a case report and literature review.

BACKGROUND: Both Caroli disease (CD) and autosomal recessive polycystic kidney disease (ARPKD) are autosomal recessive disorders, which are more commonly found in infants and children, for whom surviving to adulthood is rare. Early diagnosis and intervention can improve the survival rate to some extent. This study adopted the case of a 26-year-old pregnant woman to explore the clinical and imaging manifestations and progress of CD concomitant with ARPKD to enable a better understanding of the disease. CASE PRESENTATION: A 26-year-old pregnant woman was admitted to our hospital for more than 2 months following the discovery of pancytopenia and increased creatinine. Ultrasonography detected an enlarged left liver lobe, widened hepatic portal vein, splenomegaly, and dilated splenic vein. In addition, both kidneys were obviously enlarged and sonolucent areas of varying sizes were visible, but color Doppler flow imaging revealed no abnormal blood flow signals. The gestational age was approximately 25 weeks, which was consistent with the actual fetal age. Polyhydramnios was detected but no other abnormalities were identified. Magnetic resonance imaging revealed that the liver was plump, and polycystic liver disease was observed near the top of the diaphragm. The T1 and T2 weighted images were the low and high signals, respectively. The bile duct was slightly dilated; the portal vein was widened; and the spleen volume was enlarged. Moreover, the volume of both kidneys had increased to an abnormal shape, with multiple, long, roundish T1 and T2 abnormal signals being observed. Magnetic resonance cholangiopancreatography revealed that intrahepatic cystic lesions were connected with intrahepatic bile ducts. The patient underwent a genetic testing, the result showed she carried two heterozygous mutations in PKHD1. The patient was finally diagnosed with CD with concomitant ARPKD. The baby underwent a genetic test three months after birth, the result showed that the patient carried one heterozygous mutations in PKHD1, which indicated the baby was a PKHD1 carrier. CONCLUSIONS: This case demonstrates that imaging examinations are of great significance for the diagnosis and evaluation of CD with concomitant ARPKD.

Sigma-1 Receptor Agonist TS-157 Improves Motor Functional Recovery by Promoting Neurite Outgrowth and pERK in Rats with Focal Cerebral Ischemia.

Sigma-1 (σ-1) receptor agonists are considered as potential treatment for stroke. TS-157 is an alkoxyisoxazole-based σ-1 receptor agonist previously discovered in our group. The present study describes TS-157 profile in a battery of tests for cerebral ischemia. Initial evaluation demonstrated the compound's safety profile and blood-brain barrier permeability, as well as its ability to induce neurite outgrowth in vitro. The neurite outgrowth was shown to be mediated via σ-1 receptor agonism and involves upregulation of ERK phosphorylation (pERK). In particular, TS-157 also significantly accelerated the recovery of motor function in rats with transient middle cerebral artery occlusion (tMCAO). Overall, the results herein support the notion that σ-1 receptor agonists are potential therapeutics for stroke and further animal efficacy studies are warranted.

Synthesis and anticancer activity of novel 9,13-disubstituted berberine derivatives.

Novel berberine derivatives with disubstituents on positions C9 and C13 were synthesized and evaluated for antiproliferative activities against human prostate cancer cell lines (PC3 and DU145), breast cancer cell line (MDA-MB-231) and human colon cancer cell lines (HT29 and HCT116). All compounds showed significantly enhanced antiproliferative activities compared with berberine. Notably, compound 18e exhibited the strongest cytotoxicity against PC3 cells with an IC50 value of 0.19 µM, and the highest selectivity index (SIPC3 > 20). Further studies showed that 18e could arrest the cell cycle at G1 phase, and significantly inhibit tumor cell colony forming and migration even at low concentrations. Interestingly, 18e could significantly induce cytoplasmic vacuolation, suggesting a different mode of action from berberine.

Design, Synthesis, and In Vitro Evaluation of Benzofuro[3,2-c]Quinoline Derivatives as Potential Antileukemia Agents.

Herein, we design and synthesize an array of benzofuro[3,2-c]quinolines starting from 3-(2-methoxyphenyl)quinolin-4(1H)ones via a sequential chlorination/demethylation, intramolecular cyclization pathway. This sequential transformation was efficient, conducted under metal-free and mild reaction conditions, and yielded corresponding benzofuro[3,2-c]quinolines in high yields. In vitro biological evaluation indicated that such type of compounds showed excellent antileukemia activity and selectivity, and therefore may offer a promising hit compound for developing antileukemia compounds.

Natural continuous influent nitrifier immigration effects on nitrification and the microbial community of activated sludge systems.

Two sequencing batch reactors (SBRs) were operated for 100days under aerobic conditions, with one being fed with unsterilized municipal wastewater (USBR), and the other fed with sterilized municipal wastewater (SSBR). Respirometric assays and fluorescence in situ hybridization (FISH) results show that active nitrifiers were present in the unsterilized influent municipal wastewater. The maximum ammonia utilization rate (AUR) and nitrite utilization rate (NUR) of the unsterilized influent were 0.32±0.12mg NH4+-N/(L·hr) and 0.71±0.18mg NO2--N/(L·hr). Based on the maximum utilization rates, the estimated seeding intensity for the ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) of the USBR was 0.08g AOB/(g AOB·day) and 0.20g NOB/(g NOB·day) respectively. The fraction of nitrifiers/total bacteria in the influent was 5.35%±2.1%, the dominant AOB was Nitrosomonas spp., Nitrosococcus mobilis hybridizated with Nsm156, and the dominant NOB was Nitrospira hybridizated with Ntspa662. The influent nitrifiers potentially seeded the activated sludge of the bioreactor and hence demonstrated a mitigation of the acclimatization times and instability during start-up and early operation. The AUR and NUR in the USBR was 15% and 13% higher than the SSBR respectively during the stable stage, FISH results showed that nitrifiers population especially the Nitrospira in the USBR was higher than that in the SSBR. These results indicate that the natural continuous immigration of nitrifiers from municipal influent streams may have some repercussions on the modeling and design of bioreactors.

dCas12a:Pre-crRNA: A New Tool to Induce mRNA Degradation in Saccharomyces cerevisiae Synthetic Gene Circuits.

We describe a new way to trigger mRNA degradation in Saccharomyces cerevisiae synthetic gene circuits. Our method demands to modify either the 5'- or the 3'-UTR that flanks a target gene with elements from the pre-crRNA of type V Cas12a proteins and expresses a DNase-deficient Cas12a (dCas12a). dCas12a recognizes and cleaves the pre-crRNA motifs on mRNA sequences. Our tool does not require complex engineering operations and permits an efficient control of protein expression via mRNA degradation.

Laccase is a multitasking protein for synthetic gene circuits in the yeast Saccharomycescerevisiae.

Laccase is a multicopper oxidase enzyme that oxidizes a variety of substrates, including polyphenols and polycyclic aromatic hydrocarbons (PAHs). It catalyzes the four-electron reduction of molecular oxygen that results in the production of water as a by-product. Thus, laccase can play an important role in environmental care. Previously, we have successfully expressed Trametes trogii laccase (TtLcc1) in the yeast Saccharomyces cerevisiae. In this work, we have expressed in yeast another laccase, LacA from Trametes sp. AH28-2, and tested its function on PAHs. Yeast cells engineered to produce the two laccases performed efficient PAH degradation. Both TtLcc1 and LacA led to the construction of spatiotemporal fluorescence-pulse generators when combined with a benzoate/salicylate yeast biosensor in a two-population system. Moreover, laccases returned a visual output signal in yeast synthetic circuits-upon reacting with ABTS (2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid)). Thus, in S. cerevisiae, laccases are a powerful alternative to fluorescent reporter proteins.

Clinical Characteristics of Chlamydia psittaci Infection Diagnosed by Metagenomic Next-Generation Sequencing: A Retrospective Multi-Center Study in Fujian, China.

Objective: This study aimed to describe and compare the epidemiological, demographic, clinical, laboratory and radiological characteristics as well as the complications, treatments, and outcomes of these patients. Methods: We retrospectively investigated clinical data of patients with C. psittaci infection (psittacosis) in eight Grade IIIA hospitals of Fujian. Metagenomic next-generation sequencing (mNGS) was used identify C. psittaci in clinical samples of all included patients. Results: A total of 74 patients (39 severe/35 non-severe) was diagnosed with psittacosis, 25 (33.8%) of whom had history of poultry exposure. Common symptoms included high fever (98% [37/74]), fatigue (52.7% [39/74]), and dyspnea (51.4% [38/74]). Common manifestations in imaging included consolidation (89.2%), pleural effusion (77.0%), and air bronchogram (66.2%). Common complications included acute respiratory distress syndrome (55.4% [41/74]), type I respiratory failure (52.7% [39/74]), acute liver injury (41.9% [31/74]), and secondary infection (27.0% [20/74]). The in-hospital mortality rate was 8.11% (6/74). Conclusion: C. psittaci infection is represents an underestimated cause of CAP. For SCAP patients with poultry and bird contact history, specimens were encouraged to be sended for mNGS test in time. C. psittaci infection can lead to severe, multiple system involvement, and several complications. mNGS facilitate timely diagnosis of C. psittaci infection.

Novel small-molecule AMPK activator orally exerts beneficial effects on diabetic db/db mice.

AMP-activated protein kinase (AMPK), which is a pivotal guardian of whole-body energy metabolism, has become an attractive therapeutic target for metabolic syndrome. Previously, using a homogeneous scintillation proximity assay, we identified the small-molecule AMPK activator C24 from an optimization based on the original allosteric activator PT1. In this paper, the AMPK activation mechanism of C24 and its potential beneficial effects on glucose and lipid metabolism on db/db mice were investigated. C24 allosterically stimulated inactive AMPK α subunit truncations and activated AMPK heterotrimers by antagonizing autoinhibition. In primary hepatocytes, C24 increased the phosphorylation of AMPK downstream target acetyl-CoA carboxylase dose-dependently without changing intracellular AMP/ATP ratio, indicating its allosteric activation in cells. Through activating AMPK, C24 decreased glucose output by down-regulating mRNA levels of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) in primary hepatocytes. C24 also decreased the triglyceride and cholesterol contents in HepG2 cells. Due to its improved bioavailability, chronic oral treatment with multiple doses of C24 significantly reduced blood glucose and lipid levels in plasma, and improved the glucose tolerance of diabetic db/db mice. The hepatic transcriptional levels of PEPCK and G6Pase were reduced. These results demonstrate that this orally effective activator of AMPK represents a novel approach to the treatment of metabolic syndrome.

Extracellular polymeric substances trigger microbial immigration from partial denitrification (PD) to anammox biofilms in a long-term operated PD/anammox process in low-strength wastewater.

The immigration of microbial communities in a synergistic partial denitrification/anammox (SPDA) system was investigated in a moving bed biofilm reactor (MBBR) inoculated with partial denitrification (PD) and anaerobic ammonium oxidation (anammox) biofilms. The SPDA system was operated at 25 ± 1 °C over 260 days. The total nitrogen (TN) of the effluent was only 3.71 ± 0.92 mg·L-1 in the stable phase with a TN removal efficiency of 95.23%. The anammox process was the dominant nitrogen removal pathway with an average contribution of 74.31% to TN removal. The results of the in situ activity and key enzymatic activity revealed that the nitrate-reducing bacteria tended to immigrate to anammox biofilms. Correspondingly, the abundance of the genus Thauera, the second most dominant bacteria in anammox biofilms, quickly increased from 0.78 to 10.69% on day 50 and eventually to 16.45% on day 221 according to the Illumina MiSeq sequencing data. The microbial immigration might be caused by different extracellular polymeric substance (EPS)-mediated mechanisms in PD and anammox biofilms. For fast-growing denitrifiers, PD biofilms tend to increase the ability of mass transfer by excreting more polysaccharides to form loosely-bound EPS at the expense of the ability to harbor the nitrate-reducing bacteria. However, for the slow-growing anaerobic ammonium oxidizing bacteria (AnAOB), the anammox biofilms tend to increase the retention of AnAOB by excreting more proteins to form enhanced tightly-bound EPS at the expense of the mass transfer ability, thereby causing the detached nitrate-reducing bacteria to immigrate into anammox biofilms.