Rapid screening of point mutations by mismatch amplification mutation assay PCR.

Metabolic engineering frequently makes use of point mutation and saturation mutation library creation. At present, sequencing is the only reliable and direct technique to detect point mutation and screen saturation mutation library. In this study, mismatch amplification mutation assay (MAMA) PCR was used to detect point mutation and screen saturation mutation library. In order to fine-tune the expression of odhA encoding 2-oxoglutarate dehydrogenase E1 component, a saturating mutant library of the RBS of odhA was created in Corynebacterium glutamicum P12 based on the CRISPR-Cas2a genome editing system, which increased the L-proline production by 81.3%. MAMA PCR was used to filter out 42% of the non-mutant transformants in the mutant library, which effectively reduced the workload of the subsequent fermentation test and the number of sequenced samples. The rapid and sensitive MAMA-PCR method established in this study provides a general strategy for detecting point mutations and improving the efficiency of mutation library screening. KEY POINTS: • MAMA PCR was optimized and developed to detect point mutation. • MAMA PCR greatly improves the screening efficiency of point mutation. • Attenuation of odhA expression in P12 effectively improves proline production.

Depressive Symptoms Are Associated With Color Vision but not Olfactory Function in Patients With Parkinson's Disease.

Depressive symptoms and sensory dysfunction, such as reduction in visual and olfactory function, are common in Parkinson's disease (PD). Previous studies have suggested that depressive symptoms are associated with visual impairments and potentially with hyposmia in several types of mood disorders. However, the relationship between depressive symptoms and sensory dysfunction remains unclear in PD. To examine the association of depressive symptoms with color vision and olfactory function in PD, the authors conducted a cross-sectional study in 159 patients with PD. Depressive symptoms were measured with the Beck Depression Inventory-II (BDI-II) and the 30-item Geriatric Depression Scale (GDS-30); color vision was tested with the Farnsworth-Munsell 100 Hue Test (FMT); and olfactory function was tested with the Sniffin' Sticks Screening 12 Test. Results showed that the total error score (TES) for the FMT was significantly and independently correlated with scores on both the BDI-II and GDS-30 in a positive manner, suggesting that more severe depressive symptoms are associated with poorer color vision in PD. In addition, both somatic and effective subscores for the BDI-II were correlated with the TES on the FMT, while no significant correlation was observed between total scores on the Sniffin' Sticks Screening 12 Test and BDI-II or GDS-30. The decrease in color vision but not olfactory function was found to be associated with the severity of depressive symptoms in PD patients, supporting the idea that the occurrence of depressive symptoms in PD is linked with disruption of the visual system.

Ubiquitin-specific protease 2a stabilizes MDM4 and facilitates the p53-mediated intrinsic apoptotic pathway in glioblastoma.

The mouse double minute 4 (MDM4) oncoprotein may inhibit tumorigenesis by regulating the apoptotic mediator p53. Ubiquitin-specific protease 2a (USP2a) is a deubiquitinating enzyme that protects MDM4 against degradation, so USP2-MDM4 interaction may be a key determinant of the malignant potential of human cancers. MDM4 and USP2a, as well as the MDM4-USP2a complex, were more highly expressed in glioblastoma multiforme tissue samples from patients with good prognosis compared with patients with poor prognosis. Analysis of the prognostic parameters indicated that MDM4 expression was positively correlated with an increased likelihood for survival. Compared with the poor prognosis patients, mitochondria from good prognosis glioma patients contained higher levels of both MDM4 and the proapoptotic protein p53Ser46(P). In U87MG glioma cell line, the overexpression of MDM4 enhanced ultraviolet (UV)-induced cytochrome c release and apoptosis. In contrast, MDM4 knockdown decreased mitochondrial p53Ser46(P) levels and rescued cells from UV-induced apoptosis. The expression of MDM4 and USP2a were positively correlated with each other. MDM4-USP2a complexes were found only in the cytoplasmic fraction, whereas the mitochondrial fraction contained MDM4-p53Ser46(P) and MDM4-Bcl-2 complexes. Overexpression of USP2a increased p53 and p53Ser46(P) levels in the mitochondria, whereas simultaneous MDM4 knockdown completely reversed this effect. UV-induced apoptosis was reduced by USP2a knockdown but restored by the simultaneous overexpression of MDM4. This apoptotic response was reduced by knockdown of p53 but not p21. Our results suggest that USP2a binds to and stabilizes MDM4; thus in turn, it enhances the mitochondrial localization of p53 and promotes apoptosis in glioma cells.

Efficacy and safety of trifluridine/tipiracil plus bevacizumab versus trifluridine/tipiracil monotherapy for refractory metastatic colorectal cancer: a retrospective cohort study.

Background: Several studies demonstrated trifluridine/tipiracil (TAS-102) plus bevacizumab (BEV) had better efficacy than the monotherapy of TAS-102 in refractory metastatic colorectal cancer (mCRC). However, it remains unclear whether Chinese population can benefit from this combination or not. Hence, we conducted this retrospective cohort study to compare the efficacy and safety between TAS-102 plus BEV with TAS-102 monotherapy in refractory mCRC. Methods: This retrospective cohort study enrolled patients (any age) with refractory mCRC from Hunan Cancer Hospital. The main inclusion criteria were histopathologically and/or radiographically confirmed refractory mCRC, World Health Organization (WHO) performance status of 0 to 2, adequate organ function, and initial treatment of TAS-102 with or without BEV between November 2020 and October 2022. Previous therapy with fruquintinib or regorafenib was allowed but not mandatory. Baseline demographic and clinical characteristics were collected appropriately. Every 2 or 3 treatment cycles, the patients were assessed by computed tomography (CT) scans and clinical assessments until disease progression or loss to follow-up. The National Cancer Institute Common Terminology Criteria for Adverse Events 5.0 (NCI-CTCAE 5.0) were presented as n (%). The primary endpoint was investigator-evaluated overall survival (OS). As this is a retrospective cohort study, sample size calculation was not performed. Eligible patients would be enrolled as many as possible. Results: A total of 90 patients were enrolled, including 58 patients who received TAS-102 plus BEV and another 32 patients who received TAS-102 monotherapy. The known baseline characteristics were comparable (P<0.05). With a median follow-up of 4.60 months (range, 0.20-22.80), the median OS (mOS) time in the TAS-102 plus BEV group was longer than that in the TAS-102 monotherapy group (10.83 vs. 7.43 months), but the difference was not significant (P=0.79). The median progression-free survival (mPFS) time was comparable between the two groups (4.67 vs. 4.30 months, P=0.96). Multivariate Cox regression analysis demonstrated that undergoing therapy after TAS-102 either with or without BEV was an independent risk factor for OS [hazard ratio (HR) =0.25; 95% confidence interval (CI): 0.09-0.71, P<0.01], and previous treatment with cetuximab was an independent protective factor for PFS (HR =0.17; 95% CI: 0.03-0.91, P=0.04). Of the 70 patients who were evaluated, those receiving TAS-102 plus BEV showed trend of a higher objective response rate (ORR) and disease control rate (DCR) than those who received TAS-102 monotherapy (P=0.16 and P=0.29, respectively). Adverse events (AEs) were similar between the two groups, except that the incidence of platelet count decrease (grade ≥3) was significantly higher in the TAS-102 plus BEV group. Conclusions: There was a trend in favor of the combination of BEV plus TAS-102 regarding OS and DCR, without reaching statistical significance, and it means that there was no clear advantage of one over the other in terms of efficacy. Further prospective studies are still necessary to draw a definite conclusion.

Knockdown of cyclin D1 inhibits proliferation, induces apoptosis, and attenuates the invasive capacity of human glioblastoma cells.

Elevated cyclin D1 (CCND1) in human glioblastoma correlates with poor clinical prognosis. In this study, the human glioblastoma cell lines SHG-44 and U251 were stably transfected with short hairpin RNA (shRNA) targeting cyclin D1 or with ectogenic cyclin D1 by lentivirus-mediated transfection. Glioblastoma cells overexpressing or underexpressing cyclin D1 were then examined by in vitro growth assays, apoptosis assays, cell cycle analysis, and invasion assays. Cyclin D1 knockdown in SHG-44 cells inhibited cell proliferation, induced apoptosis, and attenuated migration across Matrigel, a model of invasive capacity. Western blot analysis and quantitative reverse-transcription polymerase chain reaction (RT-PCR) revealed that cells underexpressing CCND1 exhibited decreased multidrug resistance protein 1 (MDR1) and B-cell lymphoma-2 (Bcl-2) expression, but enhanced apoptosis effector caspase-3 expression. In contrast, cyclin D1 overexpression promoted cell proliferation, attenuated apoptosis, and enhanced invasive capacity. Furthermore, cyclin D1 overexpression was associated with increased expression of MDR1 and Bcl-2, and decreased caspase-3 expression. Results using the U251 cell line confirmed the effects of CCND1-targeted shRNA and lentivirus-mediated overexpression on proliferation and apoptosis of glioblastoma cells. Overexpression of cyclin D1 enhanced the proliferation and invasive potential of human glioblastoma cells, while reducing apoptosis. The ability to suppress the malignant phenotype by downregulating cyclin D1 expression may provide a new gene therapy approach for patients with malignant glioma.

Efficacy and safety of anti-PD-1 antibody plus regorafenib in refractory microsatellite stable metastatic colorectal cancer: a retrospective single-arm cohort study.

Background: Managements for refractory proficient mismatch repair (pMMR) or microsatellite stable (MSS) metastatic colorectal cancer (mCRC) were still challenging and controversial. Our study sought to investigate the efficacy and safety of anti-programmed cell death protein 1 (anti-PD-1) antibodies plus regorafenib in refractory pMMR/MSS mCRC. Methods: We retrospectively analyzed the efficacy and safety of 103 pMMR/MSS mCRC patients treated with at least one dose of anti-PD-1 antibodies plus regorafenib (80 mg once daily for 21 days on/7 days off 28 days as a cycle) between July 2019 and June 2021 at the Hunan Cancer Hospital. All patients had previously received at least second-line treatment. The patients were evaluated by computed tomography every 2 or 3 treatment cycles until progression or being lost to follow-up. The primary end point was overall survival (OS). Results: The median follow-up period was 5.30 (range, 0.50-22.50) months. The median OS (mOS) and medical progression-free survival (mPFS) were 8.40 and 2.50 months for the entire cohort, respectively. The mOS and mPFS were 16.07 and 3.10 months in patients who received >1 cycle of anti-PD-1 antibodies and regorafenib (n=55), which were significantly longer than 4.37 and 1.11 months in those received only 1 cycle (n=48) (both P<0.001, respectively). The Cox multivariate regression analysis demonstrated that the number of cycles of regorafenib plus PD-1 and previously undergone surgery were independent risk factors for OS, whereas Sintilimab was confirmed to have a significant better PFS compared to other anti-PD-1 antibodies. Of the 55 patients who were evaluated, 7 were diagnosed with a partial response (PR) and another 16 were diagnosed with stable disease (SD), but no patient showed a complete response (CR). Thus, the objective response rate (ORR) was 12.7% and the disease control rate was 41.8%. Treatment-related adverse events (TRAEs) of grade 3 or higher occurred in 13 (12.6%) patients. Conclusions: The combination of regorafenib plus anti-PD-1 antibodies has a manageable safety profile and may improve prognosis for pMMR/MSS mCRC patients, especially those who received >1 cycle. Compared to the other anti-PD-1 antibodies, sintilimab may be more efficacious; however, further prospective studies need to be conducted to confirm our findings.

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The intercropping of legume and cereal crops could affect crop roots growth. The relationship among intercropping, root morphology and phosphorus (P) acquisition under different P levels is still unclear. With field experiments and a rhizo-box experiment, we examined the changes of yield, biomass, P acquisition and root morphology of wheat and faba bean under different planting patterns (monocropped wheat, MW; monocropped faba bean, MF; and wheat and faba bean intercropping, W//F) and different P levels. In the rhizo-box experiment, both root weight and root-shoot ratio were increased by 21.2% and 61.5%, respectively, but shoot weight was decreased by 14.6% when wheat intercropped with faba bean. Root P content and P uptake of intercropping wheat (IW) increased by 23.8% and 12.1% when compared to MW. Both shoot and root weight, root-shoot ratio, total root length, and root volume of intercropping faba bean (IF) increased by 16.5%, 47.3%, 24.0%, 3.5%, and 8.4% as compared to MF, respectively, which resulted in higher shoot and root P content and P acquisition of IF. In the field experiment, P uptake by IW decreased by 8.7% at tillering stage, but P acquisition increased by 40.6%, 19.7%, 7.8% and 12.4% at join-ting, heading, filling, and maturity stages as compared to MW. By contrast, P acquisition of IF decreased by 9.8%, 9.0% and 5.2% at flowering, podding, and maturity stages as compared to MF. Partial least squares (PLS) regression analysis showed that root surface area and total volume of wheat and root surface area of faba bean had the greatest contribution to crop P acquisition. Intercropping induced higher root volume and root surface area which resulted in higher P acquisition under low P stress. In conclusion, interspecific interaction amplified the root-soil interface zone and increased P uptake at seedling stage under low P stress, which could contribute to the intercropping advantages at later stage.

Preserved caudate function in young-onset patients with Parkinson's disease: a dual-tracer PET imaging study.

Parkinson's disease (PD) is a highly heterogeneous clinical entity. Patients with young-onset PD (YOPD) show some characteristic manifestations to late-onset PD (LOPD). The current study aimed to investigate the cerebral dopaminergic and metabolic characteristics in YOPD with positron emission tomography (PET) imaging. In our study, 103 subjects (42 YOPD and 61 LOPD patients) accepted both 11C-N-2-carbomethoxy-3-(4-fluorophenyl)-tropane (11C-CFT) and 18F-fluorodeoxyglucose (18F-FDG) cerebral PET imaging. Sixty-two patients out of 103 patients in our study completed the cognition tests. In this limited subsection, YOPD patients performed better in cognitive functioning than LOPD patients of similar disease duration. In 11C-CFT imaging, dopamine transporter binding in caudate was relatively spared in YOPD compared with lesions in putamen. In 18F-FDG PET, YOPD patients showed increased metabolism in basal ganglia relative to the healthy controls. When compared with LOPD patients, YOPD patients exhibited hypermetabolism in caudate and hypometabolism in putamen. Furthermore, the regional metabolic values in caudate correlated positively and moderately with the dopaminergic binding deficiency in caudate. The findings of this imaging study might offer new perspectives in understanding the characteristic manifestations in YOPD in light of better-preserved cognition function.

Considerations before initiating therapy in Parkinsonism: basing on the quality of life.

OBJECTIVE: Improvement of quality-of-life (QoL) has been termed as a primary objective in initiating therapy in both Parkinson's disease (PD) and multiple system atrophy Parkinsonian subtype (MSA-P). We aimed to compare the determinants of life quality in drug naïve PD and MSA-P patients. METHODS: Eighty-six drug-naïve PD patients and thirty-five drug-naïve MSA-P patients were included to explore the determinants of QoL. Demographic information, motor deficits, and non-motor symptoms were included in the clinical assessment. RESULTS: Both motor and non-motor functions were more severely impaired in the drug-naïve MSA-P patients, with higher PDQ-39 scores indicating poorer QoL. Physical discomfort and stigma were the main affected sub-domains in PD, while mobility and activity of daily life were the main affected ones in MSA-P. BECK depressive scores and UPDRS-III scores were independent variables of PDQ-39 in MSA-P patients. Age, depression, disease stages and non-motor scores were independent variables of PDQ-39 in PD patients. INTERPRETATION: Drug-naïve MSA-P patients suffered from more severe motor and non-motor disability, as well as poorer QoL. Depression and non-motor symptoms were proved to be the most critical determinants for QoL in PD, while motor function was supposed to be the major determinant for MSA-P. When initiating therapy, physicians need to focus more on motor functions in drug-naïve MSA-P patients, but on depression in PD patients.

Update on Molecular Imaging in Parkinson's Disease.

Advances in radionuclide tracers have allowed for more accurate imaging that reflects the actions of numerous neurotransmitters, energy metabolism utilization, inflammation, and pathological protein accumulation. All of these achievements in molecular brain imaging have broadened our understanding of brain function in Parkinson's disease (PD). The implementation of molecular imaging has supported more accurate PD diagnosis as well as assessment of therapeutic outcome and disease progression. Moreover, molecular imaging is well suited for the detection of preclinical or prodromal PD cases. Despite these advances, future frontiers of research in this area will focus on using multi-modalities combining positron emission tomography and magnetic resonance imaging along with causal modeling with complex algorithms.

Cognitive characteristics in Chinese non-demented PD patients based on gender difference.

BACKGROUND: Cognitive impairment is one of the non-motor symptoms in Parkinson's disease (PD). In the present study, we aim to examine the cognitive function of non-demented Parkinson's disease patients and compare the results between male and female patients as well as control groups in search of any gender effect. METHODS: Sixty PD Patients (30 males and 30 females) from the Movement Disorders Clinic at Huashan Hospital Affiliated to Fudan University were recruited to participate in the study. One hundred age and gender matched control subjects without neurological or psychiatric disorders were voluntarily recruited. The participants were administered measures of cognition in five domains including memory, language, spatial processing abilities, attention and executive function. RESULTS: PD patients attained significantly lower scores in the visual spatial function, language and attention/executive function compared with the control group. Anti-parkinsonian treated patients performed worse in Rey-copy score, Clock Drawing Test (CDT) and Verbal Fluency-City than untreated ones. In regard to gender differences, though no general cognitive differences were found in Mini-mental State Examination (MMSE), men surpassed women on Boston naming test (BNT) while women were superior on Auditory Verbal Learning Test-long (AVLT) delayed cued recall test. CONCLUSIONS: Cognitive impairments were common in PD patients even in the absence of dementia. PD patients with anti-parkinsonian medication had worse cognitive impairment than untreated patients. Genders may have different manifestations of cognitive impairment in PD patients.

Associations between cognitive impairment and motor dysfunction in Parkinson's disease.

INTRODUCTION: Numerous studies have been carried out to explore the potential association between neurologic deficits and variable clinical manifestations of Parkinson's disease (PD). The aim of our study was to investigate the association between cognitive performance and motor dysfunction in Chinese patients with PD. METHODS: Data from 96 patients with PD were obtained from the Parkinson's disease patient cohort database of Huashan Hospital. All participants underwent a comprehensive neuropsychological evaluation to assess cognitive status, that included scoring on the Mini-mental state examination (MMSE), followed by more detailed cognitive assessment on five main cognitive domains (verbal memory, nonverbal memory, visuospatial function, language and attention/executive function). Correlations between cognitive and motor scores were investigated after controlling for age, disease duration, education, and gender. RESULTS: We report a significant correlation between subdomains of cognitive impairment and motor dysfunction using analyses of the multiple linear regression. Notably, executive function and attention was significantly associated with bradykinesia and rigidity, while visuospatial function was associated with bradykinesia and tremor. CONCLUSIONS: The association between motor dysfunction and cognitive decline in PD might highlight deficits represented by a shared neurochemical pathway.

Fasudil, a Rho kinase inhibitor, promotes the autophagic degradation of A53T α-synuclein by activating the JNK 1/Bcl-2/beclin 1 pathway.

Accumulation of α-synuclein (α-syn) is pivotally implicated in the pathogenesis of Parkinson׳s disease (PD), and enhancing its clearance might be a promising strategy in PD treatment. It has recently been shown that Rho kinase (ROCK) activation is involved in many neurodegenerative diseases, and some ROCK inhibitors might promote the degradation of abnormal protein aggregates. However, it is not known if fasudil, the only ROCK inhibitor available in clinical setting, could promote the degradation of α-syn, and ameliorate the α-syn induced neurotoxicity. In this regard, we investigated the effect of fasudil on neurite injury caused by A53T α-syn overexpression and the implicated pathway it might mediate. In the current study, we found that under the condition of A53T α-syn overexpression, the neurite outgrowth decreased significantly with the increasing expression of ROCK2. Fasudil, the ROCK inhibitor, ameliorated such neurotoxicity and promoted the clearance of A53T α-syn. Its underlying mechanism was supported by that fasudil could increase the macroautophagy activation via JNK 1 and Bcl-2 phosphorylation and beclin 1/Vps34 complex formation. Taken together, fasudil might be able to provide a novel and promising strategy for PD treatment by enhancing α-syn clearance and activating the JNK 1/Bcl-2/beclin 1 pathway.

The transducible TAT-RIZ1-PR protein exerts histone methyltransferase activity and tumor-suppressive functions in human malignant meningiomas.

Malignant meningiomas are a rare meningioma subtype and tend to have post-surgical recurrence. Significant endeavors have been taken to identify functional therapeutic targets to halt the growth of this aggressive cancer. We have recently discovered that RIZ1 is downregulated in high-grade meningiomas, and RIZ1 overexpression inhibits proliferation while promoting cell apoptosis of the IOMM-Lee malignant meningioma cell line. In this report, we show that the N-terminal PR domain of RIZ1 alone possessed growth-inhibitory activity and anticancer activity in primary human meningioma cells. Interestingly, the effects seem to be dependent on differential RIZ1 protein levels. Transducible TAT-RIZ1-PR protein could also inhibit meningioma tumor growth in nude mice models. We further demonstrate that PR protein exerts histone methyltransferase activity. A microarray analysis of TAT-RIZ1-PR-treated human malignant meningioma cells reveals 969 differentially expressed genes and 848 alternative splicing exons. Moreover, c-Myc and TXNIP, two putative downstream targets of H3K9 methylation, may be involved in regulating RIZ1 tumor-suppressive effects. The reciprocal relationship between RIZ1 and c-Myc was then validated in primary meningioma cells and human tumor samples. These findings provide insights into RIZ1 tumor suppression mechanisms and suggest that TAT-RIZ1-PR protein is a potential new epigenetic therapeutic agent for advanced meningiomas.

Triterpenes possessing an unprecedented skeleton isolated from hydrolyzate of total saponins from Gynostemma pentaphyllum.

Five novel triterpenes were isolated from hydrolyzate of total saponins from Gynostemma pentaphyllum and identified as gypensapogenin A (1), gypensapogenin B (2), gypensapogenin C (3), 3-O-ß-d-glucopyranosyl-gypensapogenin D (4) and gypensapogenin D (5), two of which (1 and 2) possess unprecedented ring A. The cyclization of the side chains-3 formed five-membered cyclic ketone rings similar to ring E in 1. The 21-oic acid-21, 23-lactone was present in the side chains of 4 and 5. We also proposed the possible formation mechanisms of compounds 1-3. Compounds 1-5 were evaluated for cytotoxic activities in three cell lines including A549, U87 and Hep3B and compound 3 showed significant activities toward A549 and U87 human cancer cells (with IC 50 values at 0.11 and 0.58 µm respectively).