Dietary Supplementation of Mixed Organic Acids Improves Growth Performance, Immunity, and Antioxidant Capacity and Maintains the Intestinal Barrier of Ira Rabbits.

The aim of this study was to investigate the effects of mixed organic acids (MOAs) on growth performance, immunity, antioxidants, intestinal digestion, and barrier function in Ira rabbits. A total of 192 weaned male Ira rabbits at 35 days of age were randomly assigned to four groups with six replicates of eight rabbits each. The rabbits in the control group (CON) were fed a basal diet, and the antibiotic group (SAL) was fed a basal diet supplemented with 60 mg/kg salinomycin. The test groups were fed a basal diet supplemented with 1000 mg/kg and 2000 mg/kg MOAs (MOA1 and MOA2, respectively). The experiment lasted for 55 days. The results showed that the ADG of Ira rabbits in the SAL group and MOA1 group was higher than that in the CON group (p < 0.05). The serum IL-6 and liver MDA levels of Ira rabbits in the SAL group, MOA1 group, and MOA2 group were lower than those in the CON group (p < 0.05). In addition, sIgA levels in the jejunal mucosa of Ira rabbits in the SAL group and MOA1 group were increased compared with those in the CON group (p < 0.05). Compared with the CON group, the gene expression of IL-6 was decreased (p < 0.05) in the jejunal mucosa of Ira rabbits in the SAL, MOA1, and MOA2 groups, while the gene expression of IL-1ß tended to decrease (p = 0.077) and the IL-10 content tended to increase (p = 0.062). Moreover, the gene expression of ZO-1 in the jejunal mucosa of Ira rabbits was elevated in the MOA1 group compared with the CON group (p < 0.05). In conclusion, dietary supplementation with MOAs can improve growth performance, enhance immune function and antioxidant capacity, and maintain the intestinal barrier in weaned Ira rabbits.

Identifying changes in flood characteristics and their causes from an event-based perspective in the Central Taihu Basin.

Increasing rainstorms induced by climate change and modification in the land surface due to urbanization have greatly altered floods at different spatio-temporal scales. However, investigating flood events in urbanized plains is challenging as anthropogenic behaviors can change river flow without rainfall. In addition, while the frequency and magnitude of floods have been well examined, knowledge about variations in the rate of flood change is still limited. To fill these gaps, we proposed a scheme that focused on flood responses to rainfall to detect changes in flood characteristics in the Central Taihu Basin, a highly urbanized region in the Yangtze River Delta of China. Four characteristic metrics were adopted to summarize the flood hydrograph, including the peak, increment, rising rate, and falling rate. We then examined trends of these metrics based on the selected rainfall-flood events from ten hydrological stations during 1970-2020. Subsequently, the reduction method was used to separate the impacts of regional climate change and human activities on flood characteristics alterations. Furthermore, the importance of fifteen factors was quantified by the random forest model. We found that there is a significant upward trend in the evolution of flood characteristics, except for the increment of floods. Flood characteristics exhibit higher values when rainfall accumulates, indicating stronger responses of floods to a large amount of rainfall. The results also show that human activities dominate and impact the peak, rising rate, and falling rate of floods more than climate change. Meanwhile, although cumulative precipitation is the most important factor, flood characteristics are also susceptible to anthropogenic factors, such as land use change and hydraulic engineering construction. Our findings, which provide insights into flood event identification and enhance the understanding of regional flood changes, will serve as a reference for water resource management and flood mitigation in urbanized areas.

Network structure and stability of the river connectivity in a rapidly urbanizing region.

River connectivity, which is key to the function of the river network, is deteriorating in highly urbanized areas. While previous studies have identified changes in the structure and connectivity of river networks, few studies have described the network structure of river connectivity and revealed the effects of urbanization on network structure. To this end, we detected the network structure and stability of river connectivity from a novel perspective of complex network theory. Taking the Taihu Plain as an example, we found that 1) the node degree of the river network was moderate (2.9), and the hub and connectivity were low (0.21 and 0.19, respectively). The hub and connectivity of the river network in the Yang-Cheng-Dian-Mao (YCDM) region were better than that in Wu-Cheng-Xi-Yu (WCXY) and Hang-Jia-Hu (HJH) regions. 2) The destruction of important nodes led to a dramatic decrease in the stability of river network connectivity. The river network structures on the Taihu Plain, HJH, WCXY, and YCDM were severely damaged when the removal rate of river network nodes exceeded 5 %, 7 %, 16 %, and 22 %, respectively. 3) The spatial response of the river network to urbanization was mainly negative, especially in highly urbanized regions. The effects of urbanization on network characteristics could be sorted as node degree (45 %), hub (21 %), and connectivity (18 %). Our results would provide theoretical support for the recognition, protection, and restoration of the river network in rapidly urbanizing regions.

The anti-inflammatory effect of lutein in broilers is mediated by regulating Toll-like receptor 4/myeloid-differentiation-factor 88 signaling pathway.

The anti-inflammatory role of lutein has been widely recognized, however, the underlying mechanism is still not fully elucidated. Hence, the effects of lutein on the intestinal health and growth performance of broilers and the action of mechanism were investigated. 288 male yellow-feathered broilers (1-day old) were randomly allocated to 3 treatment groups with 8 replicates of 12 birds each, and the control group was fed a broken rice-soybean basal diet, while the test groups were fed a basal diet added with 20 mg/kg and 40 mg/kg of lutein (LU20, LU40), respectively. The feeding trial lasted for 21 d. The results showed that 40 mg/kg lutein supplementation tended to increase ADFI (P = 0.10) and ADG (P = 0.08) of broilers. Moreover, the addition of lutein caused a decreasing trend of gene expression and concentration of proinflammatory cytokines IL-1ß (P = 0.08, P = 0.10, respectively) and IL-6 (P = 0.06, P = 0.06, respectively) and also tended to decrease the gene expression of TLR4 (P = 0.09) and MyD88 (P = 0.07) while increasing gene expression and concentration of anti-inflammatory cytokines IL-4 and IL-10 (P < 0.05) in the jejunum mucosa of broilers. Additionally, lutein supplementation increased the jejunal villi height of broilers (P < 0.05) and reduced villi damage. The experiment in vitro showed that lutein treatment reduced the gene expression of IL-1ß, IL-6, and IFN-γ in chicken intestinal epithelial cells (P < 0.05). However, this effect was diminished after knock-down of TLR4 or MyD88 genes using RNAi technology. In conclusion, lutein can inhibit the expression and secretion of proinflammatory cytokines in the jejunum mucosa and promote intestinal development of broilers, and the anti-inflammatory effect may be achieved by regulating TLR4/MyD88 signaling pathway.

Analysis on the Effect of Radiofrequency Ablation and Electrocautery in the Treatment of Vaginal Intraepithelial Neoplasia.

Objective: This research intends to investigate the clinical efficacy of radiofrequency ablation and electrocautery in treating grade I or II vaginal intraepithelial neoplasia (VaIN). Methods: This is a single-center retrospective study, which collected the clinical data of 100 patients with VaIN diagnosed by colposcopy and pathological biopsy in the Gynecology and Cervical Center of Xiangzhu Branch of the Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region between January 2020 and June 2021. Patients were divided into the study group (radiofrequency ablation treatment) and the control group (electrocautery) according to differences in treatment approaches. 6- and 12-month follow-ups were performed on all patients. Gynecological examination results, liquid-based thin-layer cytology (TCT), negative conversion of human papillomavirus (HPV), curative effects, and prognosis were recorded. Results: All patients completed regular follow-ups that lasted for 6 and 12 months. The 6- and 12-month cure rates of the study group were 76.0% and 92.0%, respectively, and the data in the control group were 70.0% and 82.0%, respectively. In terms of the 6- and 12-month negative conversion rates of HPV, the data in the study group were 68.0% and 78.0%, versus 60% and 68% in the control group, respectively. The lesion duration rate showed no statistical significance between the study group (8.0%) and the control group (P > 0.05). The analysis of postoperative follow-up complications revealed that the study group had a statistically lower overall incidence of vaginal bleeding, excessive vaginal discharge, vaginal burning sensation, and decreased vaginal elasticity than the control group (8.0% vs. 24.0% P < 0.05). Conclusion: Both radiofrequency ablation and electrocautery have obvious clinical effects in patients with grade I or II VaIN, but the former contributed to fewer operative complications and a good prognosis, which deserves clinical promotion.

A New Electron Shuttling Pathway Mediated by Lipophilic Phenoxazine via the Interaction with Periplasmic and Inner Membrane Proteins of Shewanella oneidensis MR-1.

Although it has been established that electron mediators substantially promote extracellular electron transfer (EET), electron shuttling pathways are not fully understood. Here, a new electron shuttling pathway was found in the EET process by Shewanella oneidensis MR-1 with resazurin, a lipophilic electron mediator. With resazurin, the genes encoding outer-membrane cytochromes (mtrCBA and omcA) were downregulated. Although cytochrome deletion substantially reduced biocurrent generation to 1-12% of that of wild-type (WT) cells, the presence of resazurin restored biocurrent generation to 168 µA·cm-2 (ΔmtrA/omcA/mtrC), nearly equivalent to that of WT cells (194 µA·cm-2), indicating that resazurin-mediated electron transfer was not dependent on the Mtr pathway. Biocurrent generation by resazurin was much lower in ΔcymA and ΔmtrA/omcA/mtrC/fccA/cctA mutants (4 and 6 µA·cm-2) than in WT cells, indicating a key role of FccA, CctA, and CymA in this process. The effectiveness of resazurin in EET of Mtr cytochrome mutants is also supported by cyclic voltammetry, resazurin reduction kinetics, and in situ c-type cytochrome spectroscopy results. The findings demonstrated that low molecular weight, lipophilic electron acceptors, such as phenoxazine and phenazine, may facilitate electron transfer directly from periplasmic and inner membrane proteins, thus providing new insight into the roles of exogenous electron mediators in electron shuttling in natural and engineered biogeochemical systems.

Spatial dependence of floods shaped by extreme rainfall under the influence of urbanization.

Weather system are spatially and temporally dependent, and these dependencies can result in flood events with similar behaviors. While it is well known that urbanization profoundly impacts the flood generation process, much less is known about the impacts of urbanization on the spatial dependence of floods, a major determinant of flood risk severity. To this end, a scheme was proposed to detect the flood dependence variations influenced by urbanization. Based on the scheme, we found that 1) the flood dependence can be weakened with extreme rainfall increasing from a short return period to a long return period; 2) The flood dependencies can be amplified in slightly urbanized regions and mitigated in highly urbanized regions due to intensifying urbanization. In addition, the change characteristics of the flood dependencies influenced by urbanization are first identified from the perspective of network structure. We found that urbanization can significantly affect the network structure (i.e., hub and connectivity) of flood dependence, especially in highly urbanized regions. The catchments with high hub and connectivity are prone to widespread floods and should be given more attention in flood warning and control management work, which can contribute to helping defend against floods in hazard-prone areas.

Effectiveness and feasibility of self-sampling for human papillomavirus testing for internet-based cervical cancer screening.

Objectives: Worldwide, around 18.2% of cervical cancer occurred in China, mainly because of lower screening coverage and screening quality in regional disparities. To assess self-sampling for human papillomavirus (HPV) testing, combined with the internet, as a primary cervical cancer screening (CCS) method in low-resource settings, and to establish an internet-based self-sampling CCS-management model. Methods: The women who participated registered on a CCS website. We recruited 20,136 women, aged 30-59 years, from 13 provinces in China, to perform vaginal self-sampling for HPV testing as a primary CCS, based on the internet. A questionnaire was subsequently used to investigate the acceptability of self-sampling. Results: Of the 20,103 women with qualified samples, 35.80% lived in remote areas, 37.69% had never undergone CCS, 59.96% were under-screened, and the overall prevalence of a high-risk of HPV was 13.86%. Of 8,136 respondents, 95.97% of women felt that self-sampling was easy to perform, 84.61% had no discomfort when using a self-sampling brush, 62.37% women were more likely to choose self-sampling for CCS in future, and 92.53% were willing to introduce the concept to others around them. The reliability and ease of self-sampling were independent factors influencing selection of self-sampling (p < 0.05). Conclusions: The Internet-facilitated self-sampling for HPV testing and management model for cervical cancer prevention is feasible and effective. It can be used as a supplement to the conventional screening, particularly in outlying areas with few medical resources, to improve the coverage of CCS. Clinical trial registration: https://www.chictr.org.cn, identifier: ChiCTR2000032331.

Marker-Independent Food Identification Enabled by Combing Machine Learning Algorithms with Comprehensive GC × GC/TOF-MS.

Reliable methods are always greatly desired for the practice of food inspection. Currently, most food inspection techniques are mainly dependent on the identification of special components, which neglect the combination effects of different components and often lead to biased results. By using Chinese liquors as an example, we developed a new food identification method based on the combination of machine learning with GC × GC/TOF-MS. The sample preparation methods SPME and LLE were compared and optimized for producing repeatable and high-quality data. Then, two machine learning algorithms were tried, and the support vector machine (SVM) algorithm was finally chosen for its better performance. It is shown that the method performs well in identifying both the geographical origins and flavor types of Chinese liquors, with high accuracies of 91.86% and 97.67%, respectively. It is also reasonable to propose that combining machine learning with advanced chromatography could be used for other foods with complex components.

A powerful tool for near-infrared spectroscopy: Synergy adaptive moving window algorithm based on the immune support vector machine.

Traditional trial-and-error methods are time-consuming and inefficient, especially very unfriendly to inexperienced analysts, and are sometimes still used to select preprocessing methods or wavelength variables in near-infrared spectroscopy (NIR). To deal with this problem, a new optimization algorithm called synergy adaptive moving window algorithm based on the immune support vector machine (SA-MW-ISVM) is proposed in this paper. Following the principle of SA-MW-ISVM, the original problem of calibration model optimization is transformed into a mathematical optimization problem that can be processed by the proposed immune support vector machine regression algorithm. The main objective of this optimization problem is the calibration model performance; meanwhile, the constraint conditions include a reasonable spectral data value, spectral data preprocessing method, and calibration model parameters. A unique antibody structure and specific coding and decoding method are used to achieve collaborative optimization in NIR spectroscopy. The tests on four actual near-infrared datasets, including a group of gasoline and three groups of diesel fuels, have shown that the proposed SA-MW-ISVM algorithm can significantly improve the calibration performance and thus achieve accurate prediction results. In the case of gasoline, the SA-MW-ISVM algorithm can decrease the prediction error by 44.09% compared with the common benchmark partial least square (PLS). Meanwhile, in the case of diesel fuels, the SA-MW-ISVM algorithm can decrease the prediction error of cetane number, freezing temperature, and viscosity by 9.99%, 28.69%, and 43.85%, respectively, compared with the PLS. The powerful prediction performance of the SA-MW-ISVM algorithm makes it an ideal tool for modeling near-infrared spectral data or other related application fields.

A-type FHFs mediate resurgent currents through TTX-resistant voltage-gated sodium channels.

Resurgent currents (INaR) produced by voltage-gated sodium channels are required for many neurons to maintain high-frequency firing and contribute to neuronal hyperexcitability and disease pathophysiology. Here, we show, for the first time, that INaR can be reconstituted in a heterologous system by coexpression of sodium channel α-subunits and A-type fibroblast growth factor homologous factors (FHFs). Specifically, A-type FHFs induces INaR from Nav1.8, Nav1.9 tetrodotoxin (TTX)-resistant neuronal channels, and, to a lesser extent, neuronal Nav1.7 and cardiac Nav1.5 channels. Moreover, we identified the N-terminus of FHF as the critical molecule responsible for A-type FHFs-mediated INaR. Among the FHFs, FHF4A is the most important isoform for mediating Nav1.8 and Nav1.9 INaR. In nociceptive sensory neurons, FHF4A knockdown significantly reduces INaR amplitude and the percentage of neurons that generate INaR, substantially suppressing excitability. Thus, our work reveals a novel molecular mechanism underlying TTX-resistant INaR generation and provides important potential targets for pain treatment.

Distinct biofilm formation regulated by different culture media: Implications to electricity generation.

Biofilm of Shewanella oneidensis MR-1 is extensively studied as it can transform organic compounds directly into electricity. Although revealing the biofilm regulation mechanism is crucial for enhancing bio-current, studies regarding the mechanism by which the culture condition affects biofilm formation are still lacking. The biofilm formation of S. oneidensis MR-1 in two typical media with same electron donor was investigated in this study. Bio-electricity increased 1.8 times in medium with phosphate-buffered saline (PBS) than in piperazine-1,4-bisethanesulfonic acid (PIPES). Biofilm total protein has 1.5-fold of difference between two media at day 3, and biofilm structures also differed; a fluffy biofilm with curled cells was formed in medium with PBS, whereas a compact, ordered, and closely attached biofilm was formed in medium with PIPES. Transcriptome studies clarified that the expression of genes beneficial for cell aggregation [e.g., aggA (2.3 fold), bpfA (2.8 fold) and csgB (3.9 fold)] in medium with PIPES was significantly upregulated, thus provided an explanation for the specific biofilm structure. Buffer concentration was proved to be a critical factor impacted cell morphology and current generation. The maximum current density in 30 mM of PBS and PIPES is 165 and 159 µA·cm-2 respectively, but it increased to 327 and 274 µA·cm-2 in 200 mM of PBS and PIPES. This study provides new insights into the mechanism of medium-dependent biofilm regulation, which will be beneficial for developing simple and efficient strategies to enhance bio-electricity generation.

[Analysis of Rainfall Runoff Pollution and Pollution Load Estimation for Urban Communities in a Highly Urbanized Region].

The Taihu Lake plain is a highly urbanized region in China with many water-related environmental problems. Although point-source pollution has been effectively controlled by government legislation, urban surface runoff pollution is still a major issue. Different types of urban communities were selected for rainfall runoff experiments. According to the monitoring data of rainfall events, multiple methods were used to analyze the characteristics of surface runoff pollution and estimate the pollution load for different types of communities. The results indicated that surface runoff from urban communities reduced the river water quality. Certain degrees of the 'first flush' effect occurred in different types of urban communities. The surface runoff pollution in the commercial residential community was weaker than that in commercial and private residential communities; however, the first flush occurred more frequently in the commercial residential community. Holding back 30% of the surface runoff could effectively improve the runoff water quality in commercial and private residential communities as well as the commercial residential community with restaurants. In the commercial residential community, 25% of surface runoff should be held to improve runoff water quality effectively. The loads of pollutants, especially nitrogen and phosphorus, in urban communities in the Taihu Lake basin were higher than those in other regions in China. This research can assist with the reduction of surface runoff pollution in highly urbanized communities.

Fowl Adenovirus Serotype 4 Influences Arginine Metabolism to Benefit Replication.

Hydropericardium syndrome (HPS) is caused by fowl adenovirus serotype 4 (FAdV-4). HPS has caused outbreaks in Chinese populations of broiler chickens since 2015. However, little is known about the molecular mechanisms underlying HPS. In this study, we used transcriptomic analysis to screen differentially expressed genes (DEGs) in the livers of FAdV-4-infected and noninfected chicks. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the gene network associated with the arginine metabolism pathway was enriched in livers infected by FAdV-4; 10 genes were downregulated and 8 genes were upregulated in these livers when compared to noninfected livers. The DEGs identified in livers were reanalyzed by real-time fluorescence quantitative PCR (qPCR); results indicated that the mRNA levels of the DEGs concurred with the data derived from KEGG analysis. Next, we used qPCR to detect the DEGs of the arginine metabolism pathway in a hepatocellular carcinoma cell line (LMH) after infection with FAdV-4 for 24 hr; this also indicated that the mRNA levels of the DEGs concurred with that seen in the liver. We also used si-RNA oligonucleotides to knock down the mRNA levels of iNOS in LMH cells infected with FAdV-4 and found that the viral load of FAdV-4 was increased. Further investigation revealed that the addition of 240 µg/ml of arginine into the culture medium of LMH cells infected with FAdV-4 for 24 hr led to a significant increase in the mRNA levels of iNOS but a significant reduction in the viral load of FAdV-4. Therefore, our data indicated that when broiler chickens become infected with FAdV-4, the arginine metabolic pathway in the liver becomes dysfunctional and the iNOS mRNA level decreases. This will add benefit to the replication of FAdV-4 but can be inhibited by the addition of an appropriate amount of arginine.

El adenovirus del pollo serotipo 4 influye en el metabolismo de la arginina para favorecer su replicación. El síndrome de hidropericardio (HPS) es causado por el adenovirus del pollo serotipo 4 (FAdV-4). Este síndrome ha causado brotes en las poblaciones de pollo de engorde en China desde 2015. Sin embargo, se conoce poco sobre los mecanismos moleculares subyacentes al hidropericardio. En este estudio, se utilizó el análisis transcriptómico para seleccionar genes expresados en forma diferencial (DEGs) en los hígados de pollos infectados y no infectados con el adenovirus serotipo 4. El análisis mediante la Enciclopedia de Genes and Genomas de Kyoto (KEGG) mostró que la red de genes asociada con la ruta del metabolismo de la arginina se enriqueció en hígados infectados por el adenovirus serotipo 4. Diez genes fueron regulados a la baja y ocho genes fueron regulados a la alta en estos hígados en comparación con los hígados de aves no infectadas. Los genes expresados en forma diferencial identificados en los hígados se volvieron a analizar mediante un método cuantitativo de PCR de fluorescencia en tiempo real (qPCR). Los resultados indicaron que los niveles de ARNm de los genes expresados en forma diferencial coincidían con los datos derivados del análisis la Enciclopedia de Genes and Genomas de Kyoto. Posteriormente, se utilizó qPCR para detectar los genes expresados en forma diferencial de la vía del metabolismo de la arginina en una línea celular de carcinoma hepatocelular (LMH) infectadas con el adenovirus del pollo serotipo 4 durante 24 horas. Esto también indicó que los niveles de ARNm de los genes expresados en forma diferencial coincidían con los observados en el hígado. También se utilizaron oligonucleótidos de ARN para bloquear los niveles de ARN mensajero de iNOS en células LMH infectadas con el adenovirus del pollo serotipo 4 y se descubrió que la carga viral del adenovirus aumentó. La investigación adicional reveló que la adición de 240 µg/ml de arginina en el medio de cultivo de las células LMH infectadas con el adenovirus serotipo 4 durante 24 horas condujo a un aumento significativo en los niveles de ARN mensajero de iNOS pero con una reducción significativa en la carga viral del adenovirus serotipo 4 Por lo tanto, estos datos indican que cuando los pollos de engorde se infectan con adenovirus del pollo serotipo 4, la vía metabólica de la arginina en el hígado se vuelve disfuncional y el nivel de ARN mensajero de iNOS disminuye. Esto favorecerá la replicación del adenovirus del pollo serotipo 4, pero puede inhibirse mediante la adición de una cantidad adecuada de arginina.

Repurposing Approved Drugs as Inhibitors of Kv7.1 and Nav1.8 to Treat Pitt Hopkins Syndrome.

PURPOSE: Pitt Hopkins Syndrome (PTHS) is a rare genetic disorder caused by mutations of a specific gene, transcription factor 4 (TCF4), located on chromosome 18. PTHS results in individuals that have moderate to severe intellectual disability, with most exhibiting psychomotor delay. PTHS also exhibits features of autistic spectrum disorders, which are characterized by the impaired ability to communicate and socialize. PTHS is comorbid with a higher prevalence of epileptic seizures which can be present from birth or which commonly develop in childhood. Attenuated or absent TCF4 expression results in increased translation of peripheral ion channels Kv7.1 and Nav1.8 which triggers an increase in after-hyperpolarization and altered firing properties. METHODS: We now describe a high throughput screen (HTS) of 1280 approved drugs and machine learning models developed from this data. The ion channels were expressed in either CHO (KV7.1) or HEK293 (Nav1.8) cells and the HTS used either 86Rb+ efflux (KV7.1) or a FLIPR assay (Nav1.8). RESULTS: The HTS delivered 55 inhibitors of Kv7.1 (4.2% hit rate) and 93 inhibitors of Nav1.8 (7.2% hit rate) at a screening concentration of 10 µM. These datasets also enabled us to generate and validate Bayesian machine learning models for these ion channels. We also describe a structure activity relationship for several dihydropyridine compounds as inhibitors of Nav1.8. CONCLUSIONS: This work could lead to the potential repurposing of nicardipine or other dihydropyridine calcium channel antagonists as potential treatments for PTHS acting via Nav1.8, as there are currently no approved treatments for this rare disorder.

Pain relief in a neuropathy patient by lacosamide: Proof of principle of clinical translation from patient-specific iPS cell-derived nociceptors.

BACKGROUND: Small fiber neuropathy (SFN) is a severe and disabling chronic pain syndrome with no causal and limited symptomatic treatment options. Mechanistically based individual treatment is not available. We report an in-vitro predicted individualized treatment success in one therapy-refractory Caucasian patient suffering from SFN for over ten years. METHODS: Intrinsic excitability of human induced pluripotent stem cell (iPSC) derived nociceptors from this patient and respective controls were recorded on multi-electrode (MEA) arrays, in the presence and absence of lacosamide. The patient's pain ratings were assessed by a visual analogue scale (10: worst pain, 0: no pain) and treatment effect was objectified by microneurography recordings of the patient's single nerve C-fibers. FINDINGS: We identified patient-specific changes in iPSC-derived nociceptor excitability in MEA recordings, which were reverted by the FDA-approved compound lacosamide in vitro. Using this drug for individualized treatment of this patient, the patient's pain ratings decreased from 7.5 to 1.5. Consistent with the pain relief reported by the patient, microneurography recordings of the patient's single nerve fibers mirrored a reduced spontaneous nociceptor (C-fiber) activity in the patient during lacosamide treatment. Microneurography recordings yielded an objective measurement of altered peripheral nociceptor activity following treatment. INTERPRETATION: Thus, we are here presenting one example of successful patient specific precision medicine using iPSC technology and individualized therapeutic treatment based on patient-derived sensory neurons.

Discovery of Clinical Candidate 4-[2-(5-Amino-1H-pyrazol-4-yl)-4-chlorophenoxy]-5-chloro-2-fluoro-N-1,3-thiazol-4-ylbenzenesulfonamide (PF-05089771): Design and Optimization of Diaryl Ether Aryl Sulfonamides as Selective Inhibitors of NaV1.7.

A series of acidic diaryl ether heterocyclic sulfonamides that are potent and subtype selective NaV1.7 inhibitors is described. Optimization of early lead matter focused on removal of structural alerts, improving metabolic stability and reducing cytochrome P450 inhibition driven drug-drug interaction concerns to deliver the desired balance of preclinical in vitro properties. Concerns over nonmetabolic routes of clearance, variable clearance in preclinical species, and subsequent low confidence human pharmacokinetic predictions led to the decision to conduct a human microdose study to determine clinical pharmacokinetics. The design strategies and results from preclinical PK and clinical human microdose PK data are described leading to the discovery of the first subtype selective NaV1.7 inhibitor clinical candidate PF-05089771 (34) which binds to a site in the voltage sensing domain.

Biophysical and Pharmacological Characterization of Nav1.9 Voltage Dependent Sodium Channels Stably Expressed in HEK-293 Cells.

The voltage dependent sodium channel Nav1.9, is expressed preferentially in peripheral sensory neurons and has been linked to human genetic pain disorders, which makes it target of interest for the development of new pain therapeutics. However, characterization of Nav1.9 pharmacology has been limited due in part to the historical difficulty of functionally expressing recombinant channels. Here we report the successful generation and characterization of human, mouse and rat Nav1.9 stably expressed in human HEK-293 cells. These cells exhibit slowly activating and inactivating inward sodium channel currents that have characteristics of native Nav1.9. Optimal functional expression was achieved by coexpression of Nav1.9 with ß1/ß2 subunits. While recombinantly expressed Nav1.9 was found to be sensitive to sodium channel inhibitors TC-N 1752 and tetracaine, potency was up to 100-fold less than reported for other Nav channel subtypes despite evidence to support an interaction with the canonical local anesthetic (LA) binding region on Domain 4 S6. Nav1.9 Domain 2 S6 pore domain contains a unique lysine residue (K799) which is predicted to be spatially near the local anesthetic interaction site. Mutation of this residue to the consensus asparagine (K799N) resulted in an increase in potency for tetracaine, but a decrease for TC-N 1752, suggesting that this residue can influence interaction of inhibitors with the Nav1.9 pore. In summary, we have shown that stable functional expression of Nav1.9 in the widely used HEK-293 cells is possible, which opens up opportunities to better understand channel properties and may potentially aid identification of novel Nav1.9 based pharmacotherapies.