M2 macrophages independently promote beige adipogenesis via blocking adipocyte Ets1.

Adipose tissue macrophages can promote beige adipose thermogenesis by altering local sympathetic activity. Here, we perform sympathectomy in mice and further eradicate subcutaneous adipose macrophages and discover that these macrophages have a direct beige-promoting function that is independent of sympathetic system. We further identify adipocyte Ets1 as a vital mediator in this process. The anti-inflammatory M2 macrophages suppress Ets1 expression in adipocytes, transcriptionally activate mitochondrial biogenesis, as well as suppress mitochondrial clearance, thereby increasing the mitochondrial numbers and promoting the beiging process. Male adipocyte Ets1 knock-in mice are completely cold intolerant, whereas male mice lacking Ets1 in adipocytes show enhanced energy expenditure and are resistant to metabolic disorders caused by high-fat-diet. Our findings elucidate a direct communication between M2 macrophages and adipocytes, and uncover a function for Ets1 in responding to macrophages and negatively governing mitochondrial content and beige adipocyte formation.

Adaptive modeling optimized by the data fusion strategy: Real-time dying cell percentage prediction using capacitance spectroscopy.

The previous research showcased a partial least squares (PLS) regression model accurately predicting cell death percentages using in-line capacitance spectra. The current study advances the model accuracy through adaptive modeling employing a data fusion approach. This strategy enhances prediction performance by incorporating variables from the Cole-Cole model, conductivity and its derivatives over time, and Mahalanobis distance into the predictor matrix (X-matrix). Firstly, the Cole-Cole model, a mechanistic model with parameters linked to early cell death onset, was integrated to enhance prediction performance. Secondly, the inclusion of conductivity and its derivatives over time in the X-matrix mitigated prediction fluctuations resulting from abrupt conductivity changes during process operations. Thirdly, Mahalanobis distance, depicting spectral changes relative to a reference spectrum from a previous time point, improved model adaptability to independent test sets, thereby enhancing performance. The final data fusion model substantially decreased root-mean squared error of prediction (RMSEP) by around 50%, which is a significant boost in prediction accuracy compared to the prior PLS model. Robustness against reference spectrum selection was confirmed by consistent performance across various time points. In conclusion, this study illustrates that the data fusion strategy substantially enhances the model accuracy compared to the previous model relying solely on capacitance spectra.

Automated Online-Sampling Multidimensional Liquid Chromatography with Feedback-Control Capability as a Framework for Real-Time Monitoring of mAb Critical Quality Attributes in Multiple Bioreactors.

Real-time monitoring of biopharmaceutical reactors is becoming increasingly important as the processes become more complex. During the continuous manufacturing of monoclonal antibodies (mAbs), the desired mAb product is continually created and collected over a 30 day process, where there can be changes in quality over that time. Liquid chromatography (LC) is the workhorse instrumentation capable of measuring mAb concentration as well as quality attributes such as aggregation, charge variants, oxidation, etc. However, traditional offline sampling is too infrequent to fully characterize bioprocesses, and the typical time from sample generation to data analysis and reporting can take weeks. To circumvent these limitations, an automated online sampling multidimensional workflow was developed to enable streamlined measurements of mAb concentration, aggregation, and charge variants. This analytical framework also facilitates automated data export for real-time analysis of up to six bioreactors, including feedback-controlling capability using readily available LC technology. This workflow increases the data points per bioreactor, improving the understanding of each experiment while also reducing the data turnaround time from weeks to hours. Examples of effective real-time analyses of mAb critical quality attributes are illustrated, showing substantial throughput improvements and accurate results while minimizing labor and manual intervention.

Capacitance spectroscopy enables real-time monitoring of early cell death in mammalian cell culture.

BACKGROUND/AIMS: Previous work developed a quantitative model using capacitance spectroscopy in an at-line setup to predict the dying cell percentage measured from a flow cytometer. This work aimed to transfer the at-line model to monitor lab-scale bioreactors in real-time, waiving the need for frequent sampling and enabling precise controls. METHODS AND RESULTS: Due to the difference between the at-line and in-line capacitance probes, direct application of the at-line model resulted in poor accuracy and high prediction bias. A new model with a variable range and offering similar spectral shape across all probes was first constructed, improving prediction accuracy. Moreover, the global calibration method included the variance of different probes and scales in the model, reducing prediction bias. External parameter orthogonalization, a preprocessing method, also mitigated the interference from feeding, which further improved model performance. The root-mean-square error of prediction of the final model was 6.56% (8.42% of the prediction range) with an R2 of 92.4%. CONCLUSION: The culture evolution trajectory predicted by the in-line model captured the cell death and alarmed cell death onset earlier than the trypan blue exclusion test. Additionally, the incorporation of at-line spectra following orthogonal design into the calibration set was shown to generate calibration models that are more robust than the calibration models constructed using the in-line spectra only. This is advantageous, as at-line spectral collection is easier, faster, and more material-sparing than in-line spectra collection.

NIR Spectroscopy as an Online PAT Tool for a Narrow Therapeutic Index Drug: Toward a Platform Approach Across Lab and Pilot Scales for Development of a Powder Blending Monitoring Method and Endpoint Determination.

An online near-infrared (NIR) spectroscopy platform system for real-time powder blending monitoring and blend endpoint determination was tested for a phenytoin sodium formulation. The study utilized robust experimental design and multiple sensors to investigate multivariate data acquisition, model development, and model scale-up from lab to manufacturing. The impact of the selection of various blend endpoint algorithms on predicted blend endpoint (i.e., mixing time) was explored. Spectral data collected at two process scales using two NIR spectrometers was incorporated in a single (global) calibration model. Unique endpoints were obtained with different algorithms based on standard deviation, average, and distributions of concentration prediction for major components of the formulation. Control over phenytoin sodium's distribution was considered critical due to its narrow therapeutic index nature. It was found that algorithms sensitive to deviation from target concentration offered the simplest interpretation and consistent trends. In contrast, algorithms sensitive to global homogeneity of active and excipients yielded the longest mixing time to achieve blending endpoint. However, they were potentially more sensitive to subtle uniformity variations. Qualitative algorithms using principal component analysis (PCA) of spectral data yielded the prediction of shortest mixing time for blending endpoint. The hybrid approach of combining NIR data from different scales presents several advantages. It enables simplifying the chemometrics model building process and reduces the cost of model building compared to the approach of using data solely from commercial scale. Success of such a hybrid approach depends on the spectroscopic variability captured at different scales and their relative contributions in the final NIR model.

Rapid at-line early cell death quantification using capacitance spectroscopy.

Cell death is one of the failure modes of mammalian cell culture. Apoptosis is a regulated cell death process mainly observed in cell culture. Timely detection of apoptosis onset allows opportunities for preventive controls that ensure high productivity and consistent product quality. Capacitance spectroscopy captures the apoptosis-related cellular properties changes and thus quantifies the percentage of dying cells. This study demonstrated a quantification model that measures the percentage of apoptotic cells using a capacitance spectrometer in an at-line setup. When predicting the independent test set collected from bench-scale bioreactors, the root-mean-squared error of prediction was 8.8% (equivalent to 9.9% of the prediction range). The predicted culture evolution trajectory aligned with measured values from the flow cytometer. Furthermore, this method alarms cell death onset earlier than the traditional viability test, that is, the trypan blue exclusion test. Compared to flow cytometry (the traditional early cell death detection method), this method is rapid, simple, and less labor-intensive. In addition, this at-line setup can be easily transferred between scales (e.g., lab-scale for development to manufacturing scale), which benefits process transfers between facilities, scale-up, and other process transitions.

Rapid serum-free/suspension adaptation: Medium development using a definitive screening design for Chinese hamster ovary cells.

The biopharmaceutical industry prefers to culture the mammalian cells in suspension with a serum-free media (SFM) due to improved productivity and process consistency. However, mammalian cells preferentially grow as adherent cells in a complete medium (CM) containing serum. Therefore, cells require adaptation from adherence in CM to suspension culture in SFM. This work proposes an adaptation method that includes media supplementation during the adaption of Chinese hamster ovary cells. As a result, the adaptation was accelerated compared to the traditional repetitive subculturing. Ca2+ /Mg2+ supplementation significantly reduced the doubling time compared to the adaptation without supplementation during the adaptation of adherent cells from 100% CM to 75% CM (p < 0.05). Furthermore, a definitive screening design (DSD) was applied to select essential nutrients during the adaptation from 10% CM to 0% CM. The main effects of Ca2+ and Dulbecco's modified essential medium (DMEM) were found significant to both viable cell density and viability at harvest. Additionally, the interaction term between Ca2+ and DMEM was found significant, which highlights the ability of DSD to capture interaction terms. Eventually, the media supplementation method resulted in adaptation SFM in 27 days, compared to the previously reported 66 days. Additionally, the membrane surface integrin expression was found significantly decreased when adherent cells were adapted to suspension. Moreover, the Ca2+ /Mg2+ supplementation correlated with faster integrin recovery after trypsinization. However, faster integrin recovery did not contribute to the accelerated cell growth when subculturing from 100% CM to 75% CM.

Immune gene expression profiling reveals heterogeneity in luminal breast tumors.

BACKGROUND: Heterogeneity of immune gene expression patterns of luminal breast cancer (BC), which is clinically heterogeneous and overall considered as low immunogenic, has not been well studied especially in non-European populations. Here, we aimed at characterizing the immune gene expression profile of luminal BC in an Asian population and associating it with patient characteristics and tumor genomic features. METHODS: We performed immune gene expression profiling of tumor and adjacent normal tissue in 92 luminal BC patients from Hong Kong using RNA-sequencing data and used unsupervised consensus clustering to stratify tumors. We then used luminal patients from The Cancer Genome Atlas (TCGA, N = 564) and a Korean breast cancer study (KBC, N = 112) as replication datasets. RESULTS: Based on the expression of 130 immune-related genes, luminal tumors were stratified into three distinct immune subtypes. Tumors in one subtype showed higher level of tumor-infiltrating lymphocytes (TILs), characterized by T cell gene activation, higher expression of immune checkpoint genes, higher nonsynonymous mutation burden, and higher APOBEC-signature mutations, compared with other luminal tumors. The high-TIL subtype was also associated with lower ESR1/ESR2 expression ratio and increasing body mass index. The comparison of the immune profile in tumor and matched normal tissue suggested a tumor-derived activation of specific immune responses, which was only seen in high-TIL patients. Tumors in a second subtype were characterized by increased expression of interferon-stimulated genes and enrichment for TP53 somatic mutations. The presence of three immune subtypes within luminal BC was replicated in TCGA and KBC, although the pattern was more similar in Asian populations. The germline APOBEC3B deletion polymorphism, which is prevalent in East Asian populations and was previously linked to immune activation, was not associated with immune subtypes in our study. This result does not support the hypothesis that the germline APOBEC3B deletion polymorphism is the driving force for immune activation in breast tumors in Asian populations. CONCLUSION: Our findings suggest that immune gene expression and associated genomic features could be useful to further stratify luminal BC beyond the current luminal A/B classification and a subset of luminal BC patients may benefit from checkpoint immunotherapy, at least in Asian populations.

Urinary bisphenol A and incidence of metabolic syndrome among Chinese men: a prospective cohort study from 2013 to 2017.

OBJECTIVES: Experimental studies suggested that bisphenol A (BPA) exposure increased the risk of metabolic syndrome (MetS) through the mechanism of insulin resistance. All previous epidemiological studies of BPA and MetS were cross-sectional studies, and their findings were mixed. This study aims to provide further evidence on the association between urinary BPA and risk of MetS using a prospective cohort study in China. METHODS: The study population was from the Shenzhen Night shift workers' cohort. A total of 1227 male workers were recruited from the baseline survey in 2013 and then followed until 2017. Modified Adult Treatment Panel III criteria were used to identify the cases of MetS. Urinary BPA concentration was assessed using high-performance liquid chromatography-tandem mass spectrometry, and it was categorised into three subgroups by tertiles to obtain the adjusted HR (aHR) and 95% CI using Cox proportional hazard model. RESULTS: During 4 years of follow-up, 200 subjects developed MetS. Compared with the lowest urinary BPA subgroup, a weakly increased risk of MetS was suggested among those with the middle (aHR=1.19, 95% CI 0.87 to 1.63) and high level of urinary BPA (aHR=1.16, 95% CI 0.84 to 1.59); however, the significant association with MetS was restricted primarily to the smokers, showing a positive gradient with urinary BPA (middle level: aHR=2.40, 95% CI 1.13 to 5.08; high level: aHR=2.87, 95% CI 1.38 to 5.98; p trend=0.010). CONCLUSION: This prospective cohort study provided further evidence that exposure to BPA may increase the risk of MetS, and this association was further positively modified by cigarette smoking.

Urinary parabens in adults from South China: Implications for human exposure and health risks.

Parabens are a kind of preservatives widely used in cosmetic and personal care products and ubiquitously detected in the environment. However, little is known on human exposure to these chemicals. Our study mainly investigated the urinary parabens in adults from South China to evaluate the cumulative risk of paraben exposure. A total of 562 urine samples were collected from adult workers for the determination of methyl paraben (MeP), ethyl paraben (EtP), propyl paraben (PrP), butyl paraben, and benzyl parabens. High detection frequencies (≥98%) were observed for MeP, EtP, and PrP with median concentrations of 8.88, 5.11, and 1.44 µg/L, respectively. Urinary parabens was 4.5-46.2 fold higher in urine of females than those in males. Urinary MeP was associated with alcohol drinking and a history of tumor, while urinary PrP was negatively associated with education levels of the subjects. There were not significant associations between urinary concentrations of parabens and body mass index, which indicated that obesity was not associated with paraben exposure. Also, parabens did not correlate with human dietary habits. Although the total estimated daily intake (TEDI) of the major compound MeP and EtP in adult workers was lower than the acceptable daily intake (ADI), the TEDI of PrP exceed the ADI for a very few subjects, especially for females and low-educated ones, suggesting potential health risks.

Feedforward and Feedback Control of a Pharmaceutical Coating Process.

This work demonstrates the use of a combination of feedforward and feedback loops to control the controlled release coating of theophylline granules. Feedforward models are based on the size distribution of incoming granules and are used to set values for the airflow in the fluid bed processor and the target coat weight to be applied to the granules. The target coat weight of the granules is controlled by a feedback loop using NIR spectroscopy to monitor the progress of the process. By combining feedforward and feedback loops, significant variation in the size distributions and ambient conditions were accommodated in the fluid bed coating of the granules and a desired dissolution profile was achieved. The feedforward component of the control system was specifically tested by comparing the performance of the control system with and without this element by Monte Carlo simulation.

Night shift work and abnormal liver function: is non-alcohol fatty liver a necessary mediator?

OBJECTIVES: Accumulated evidence implies that night shift work may trigger liver dysfunction. Non-alcoholic fatty liver (NAFL) is suggested to be a necessary mediator in this process. This study aimed to examine the relationship between night shift work and elevated level of alanine transaminase (e-ALT) of workers and investigate the potential mediation effect of NAFL. METHODS: This study included all male workers from the baseline survey of a cohort of night shift workers. Information on demographics, lifestyle and lifetime working schedule was collected by face-to-face interview. Liver sonography was used to identify NAFL cases. Serum ALT level was detected by an automatic biochemical analyser. e-ALT was defined as ALT >40 U/L. Logistic regression models were used to evaluate ORs, and mediation analysis was employed to examine the mediation effect. RESULTS: Among 4740 male workers, 39.5% were night shift workers. Night shift workers had an increased risk of e-ALT (OR, 1.19, 95% CI 1.00 to 1.42). With the increase in night shift years, the OR of e-ALT increased from 1.03 (95% CI 0.77 to 1.36) to 1.60 (95% CI 1.08 to 2.39) among workers without NAFL. A similar trend was not found among workers with NAFL. In addition, no significant mediation effect of NAFL in the association between night shift work and e-ALT was found. CONCLUSIONS: Night shift work is positively associated with abnormal liver function, in particular among workers without NAFL. Shift work involving circadian disruption is likely to exert a direct effect on liver dysfunction rather than rely on the mediation effect of NAFL.

Dietary acrylamide exposure was associated with mild cognition decline among non-smoking Chinese elderly men.

The aim of the study is to explore the longitudinal association of dietary acrylamide exposure with cognitive performance in Chinese elderly. The analysis was conducted among 2534 non-smoking elderly men and women based on a prospective study, Mr. and Ms. OS Hong Kong. Dietary acrylamide intake was assessed by food frequency questionnaires with data on local food contamination, derived from the first Hong Kong Total Diet Study. Global cognitive function was assessed by Cantonese version of Mini-Mental State Exam (MMSE) at the baseline and the 4th year of follow-up. Multivariable-adjusted linear and logistic regression models were used to assess the associations of dietary acrylamide with MMSE score changes or risk of poor cognition. The results indicated that among men with MMSE ≥ 18, each one SD increase of acrylamide decreased MMSE score by 7.698% (95%CI: -14.943%, -0.452%; p = 0.037). Logistic regression revealed an increased risk of poor cognition (MMSE ≤ 26) in men with HR of 3.356 (1.064~10.591, p = 0.039). The association became non-significance after further adjustment for telomere length. No significant association was observed in women. Dietary acrylamide exposure was associated with a mild cognitive decline or increased risk of poor cognition over a 4-year period in non-smoking Chinese elderly men.

Dietary acrylamide exposure was associated with increased cancer mortality in Chinese elderly men and women: a 11-year prospective study of Mr. and Ms. OS Hong Kong.

AIM: Our study aims to investigate the association between dietary acrylamide exposure and cancer mortality among Chinese elderly. METHODS: A prospective cohort of 4000 elderly men and women aged 65 years and above (Mr. and Ms. OS Hong Kong study) was recruited from local communities from 2001 to 2003. Dietary exposure to acrylamide was evaluated at baseline based on a validated food frequency questionnaire and an acrylamide database from the 1st Hong Kong Total Diet Study. Data on mortality statistics through March 2014 were obtained from the Death Registry of the Department of Health of Hong Kong with a median follow-up of 11.1 years. Cox proportional hazards models were used to examine the association between the acrylamide exposure and cancer mortality. Sex hormones were assessed in men. RESULTS: During a median follow-up of 11.1 years (39,271 person-years), we ascertained 330 cancer deaths. Vegetables (43.7%) and cereals (28.9%) products were the major contributors to dietary acrylamide. Compared with the lowest quartile of acrylamide intake (<9.9 µg/day), the multivariable hazard ratios for the highest quartile (>17.1 µg/day) were 1.9 (95% CI 1.3-2.8; P trend < 0.01), 1.9 (95% CI 1.0-3.6; P trend = 0.05), and 2.0 (95% CI 1.0-4.0; P trend = 0.06) for the cancer mortality from overall, digestive and respiratory system, respectively. The associations were attenuated to null after further adjustment for circulating free estradiol in men. No statistically significant interactions were observed between acrylamide exposure and sex, obesity and overall lifestyle pattern scores. CONCLUSIONS: The longitudinal data provided evidence that dietary acrylamide, in amounts that Chinese elderly are typically exposed to, was associated with increased cancer mortality. Circulating free estradiol may mediate the association in men.

Impact of formulation and process variables on solid-state stability of theophylline in controlled release formulations.

Understanding the impact of pharmaceutical processing, formulation excipients and their interactions on the solid-state transitions of pharmaceutical solids during use and in storage is critical in ensuring consistent product performance. This study reports the effect of polymer viscosity, diluent type, granulation and granulating fluid (water and isopropanol) on the pseudopolymorphic transition of theophylline anhydrous (THA) in controlled release formulations as well as the implications of this transition on critical quality attributes of the tablets. Accordingly, 12 formulations were prepared using a full factorial screening design and monitored over a 3 month period at 40 °C and 75%. Physicochemical characterization revealed a drastic drop in tablet hardness accompanied by a very significant increase in moisture content and swelling of all formulations. Spectroscopic analysis (ssNMR, Raman, NIR and PXRD) indicated conversion of THA to theophylline monohydrate (TMO) in all formulations prepared by aqueous wet granulation in as early as two weeks. Although all freshly prepared formulations contained THA, the hydration-dehydration process induced during aqueous wet granulation hastened the pseudopolymorphic conversion of theophylline during storage through a cascade of events. On the other hand, no solid state transformation was observed in directly compressed formulations and formulations in which isopropanol was employed as a granulating fluid even after the twelve weeks study period. The transition of THA to TMO resulted in a decrease in dissolution while an increase in dissolution was observed in directly compressed and IPA granulated formulation. Consequently, the impact of pseudopolymorphic transition of theophylline on dissolution in controlled release formulations may be the net result of two opposing factors: swelling and softening of the tablets which tend to favor an increase in drug dissolution and hydration of theophylline which decreases the drug dissolution.