More than a feeling: Scalp EEG and eye signals in conscious tactile perception.

Understanding the neural basis of consciousness is a fundamental goal of neuroscience, and sensory perception is often used as a proxy for consciousness in empirical studies. However, most studies rely on reported perception of visual stimuli. Here we present behavior, high density scalp EEG and eye metric recordings collected simultaneously during a novel tactile threshold perception task. We found significant N80, N140 and P300 event related potentials in perceived trials and in perceived versus not perceived trials. Significance was limited to a P100 and P300 in not perceived trials. We also found an increase in pupil diameter and blink rate and a decrease in microsaccade rate following perceived relative to not perceived tactile stimuli. These findings support the use of eye metrics as a measure of physiological arousal associated with conscious perception. Eye metrics may also represent a novel path toward the creation of tactile no-report tasks in the future.

Early cortical signals in visual stimulus detection.

During visual conscious perception, the earliest responses linked to signal detection are little known. The current study aims to reveal the cortical neural activity changes in the earliest stages of conscious perception using recordings from intracranial electrodes. Epilepsy patients (N=158) were recruited from a multi-center collaboration and completed a visual word recall task. Broadband gamma activity (40-115Hz) was extracted with a band-pass filter and gamma power was calculated across subjects on a common brain surface. Our results show early gamma power increases within 0-50ms after stimulus onset in bilateral visual processing cortex, right frontal cortex (frontal eye fields, ventral medial/frontopolar, orbital frontal) and bilateral medial temporal cortex regardless of whether the word was later recalled. At the same early times, decreases were seen in the left rostral middle frontal gyrus. At later times after stimulus onset, gamma power changes developed in multiple cortical regions. These included sustained changes in visual and other association cortical networks, and transient decreases in the default mode network most prominently at 300-650ms. In agreement with prior work in this verbal memory task, we also saw greater increases in visual and medial temporal regions as well as prominent later (> 300ms) increases in left hemisphere language areas for recalled versus not recalled stimuli. These results suggest an early signal detection network in the frontal, medial temporal, and visual cortex is engaged at the earliest stages of conscious visual perception.

Optimal initial antibiotic regimen for the treatment of acute appendicitis: a systematic review and network meta-analysis with surgical intervention as the common comparator.

BACKGROUND: The optimal antibiotic regimen for the medical management of acute appendicitis remains unknown due to a lack of head-to-head comparisons between different antibiotic regimens. METHODS: We systematically searched the PubMed, EMBASE, Scopus and Cochrane Central Register of Controlled Trials databases from their inception through to August 2020. We selected randomized controlled trials (RCTs) or observational studies comparing antibiotic therapy and appendectomy as the initial treatment for adult or paediatric patients with acute appendicitis. We performed a Bayesian network meta-analysis (NMA) to obtain the indirect comparison results between different antibiotic regimens by employing the group managed by surgery as a common comparator. Antibiotic regimens were classified into three categories: those including a carbapenem; those including a cephalosporin; and those including a ß-lactam/ß-lactamase inhibitor combination. RESULTS: A total of 9 RCTs (adults, n = 8; paediatrics, n = 1) and 12 observational studies (adults, n = 3; paediatrics, n = 9) were included in the NMA, with a total of 4551 patients. The most commonly administered regimen was a ß-lactam/ß-lactamase inhibitor combination (9/21; 43%), followed by a cephalosporin (7/21; 33%) or a carbapenem (5/21; 24%). The NMA indicated that surgery significantly increased 1 year treatment success, compared with cephalosporins [OR: 16.79; 95% credible interval: 3.8-127.64] or ß-lactam/ß-lactamase inhibitor combinations (OR: 19.99; 95% credible interval: 4.87-187.57), but not carbapenems (OR: 3.50, 95% credible interval: 0.55-38.63). In contrast, carbapenems were associated with fewer treatment-related complications compared with surgery (OR: 0.12; 95% credible interval: 0.01-0.85). CONCLUSIONS: Carbapenems might be recommended as the initial antibiotic regimen for the non-operative management of adult patients with acute appendicitis. Nevertheless, due to the imprecise estimates in our NMA, additional RCTs are needed to corroborate these findings, especially for paediatric patients.

Process analytical technology for on-line monitoring of quality attributes during single-use ultrafiltration/diafiltration.

Process analytical technology (PAT) is a fast-growing field within bioprocessing that enables innovation in biological drug manufacturing. This study demonstrates novel PAT methods for monitoring multiple quality attributes simultaneously during the ultrafiltration and diafiltration (UF/DF) process operation, the final step of monoclonal antibody (mAb) purification. Size exclusion chromatography (SEC) methods were developed to measure excipients arginine, histidine, and high molecular weight (HMW) species using a liquid chromatography (LC) system with autosampler for both on-line and at-line PAT modes. The methods were applied in UF/DF studies for the comparison of single-use tangential flow filtration (TFF) cassettes to standard reusable cassettes to achieve very high concentration mAb drug substance (DS) in the order of 100-200 g/L. These case studies demonstrated that single-use TFF cassettes are a functionally equivalent, low-cost alternative to standard reusable cassettes, and that the on-line PAT measurement of purity and excipient concentration was comparable to orthogonal offline methods. These PAT applications using an on-line LC system equipped with onboard sample dilution can become a platform system for monitoring of multiple attributes over a wide dynamic range, a potentially valuable tool for biological drug development and manufacturing.

Productivity improvement and charge variant modulation for intensified cell culture processes by adding a carboxypeptidase B (CpB) treatment step.

The goal of cell culture process intensification is to improve productivity while maintaining acceptable quality attributes. In this report, four processes, namely a conventional manufacturing Process A, and processes intensified by enriched N-1 seed (Process B), by perfusion N-1 seed (Process C), and by perfusion production (Process D) were developed for the production of a monoclonal antibody. The three intensified processes substantially improved productivity, however, the product either failed to meet the specification for charge variant species (main peak) for Process D or the production process required early harvest to meet the specification for charge variant species, Day 10 or Day 6 for Processes B and C, respectively. The lower main peak for the intensified processes was due to higher basic species resulting from higher C-terminal lysine. To resolve this product quality issue, we developed an enzyme treatment method by introducing carboxypeptidase B (CpB) to clip the C-terminal lysine, leading to significantly increased main peak and an acceptable and more homogenous product quality for all the intensified processes. Additionally, Processes B and C with CpB treatment extended bioreactor durations to Day 14 increasing titer by 38% and 108%, respectively. This simple yet effective enzyme treatment strategy could be applicable to other processes that have similar product quality issues.

Online monitoring and control of upstream cell culture process using 1D and 2D-LC with SegFlow interface.

The biopharmaceutical industry is transitioning from currently deployed batch-mode bioprocessing to a highly efficient and agile next-generation bioprocessing with the adaptation of continuous bioprocessing, which reduces capital investment and operational costs. Continuous bioprocessing, aligned with FDA's quality-by-design platform, is designed to develop robust processes to deliver safe and effective drugs. With the deployment of knowledge-based operations, product quality can be built into the process to achieve desired critical quality attributes (CQAs) with reduced variability. To facilitate next-generation continuous bioprocessing, it is essential to embrace a fundamental shift-in-paradigm from "quality-by-testing" to "quality-by-design," which requires the deployment of process analytical technologies (PAT). With the adaptation of PAT, a systematic approach of process and product understanding and timely process control are feasible. Deployment of PAT tools for real-time monitoring of CQAs and feedback control is critical for continuous bioprocessing. Given the current deficiency in PAT tools to support continuous bioprocessing, we have integrated Infinity 2D-LC with a post-flow-splitter in conjunction with the SegFlow autosampler to the bioreactors. With this integrated system, we have established a platform for online measurements of titer and CQAs of monoclonal antibodies as well as amino acid analysis of bioreactor cell culture.

Understanding the pathway and kinetics of aspartic acid isomerization in peptide mapping methods for monoclonal antibodies.

Isomerization of aspartic acid (Asp) in therapeutic proteins could lead to safety and efficacy concerns. Thus, accurate quantitation of various Asp isomerization along with kinetic understanding of the variant formations is needed to ensure optimal process development and sufficient product quality control. In this study, we first observed Asp-succinimide conversion in complementarity-determining regions (CDRs) Asp-Gly motif of a recombinant mAb through ion exchange chromatography, intact protein analysis by mass spectrometry, and LC-MS/MS. Then, we developed a specific peptide mapping method, with optimized sample digestion conditions, to accurately quantitate Asp-succinimide-isoAsp variants at peptide level without method-induced isomerization. Various kinetics of Asp-succinimide-isoAsp isomerization pathways were elucidated using 18O labeling followed by LC-MS analysis. Molecular modeling and molecular dynamic simulation provide additional insight on the kinetics of Asp-succinimide formation and stability of succinimide intermediate. Findings of this work shed light on the molecular construct and the kinetics of the formation of isoAsp and succinimide in peptides and proteins, which facilitates analytical method development, protein engineering, and late phase development for commercialization of therapeutic proteins.

A novel approach enables imaged capillary isoelectric focusing analysis of PEGylated proteins.

PEGylation has been used as a strategy to enhance pharmacokinetic properties of therapeutic proteins by pharmaceutical industry. Imaged CIEF (iCIEF) is the current industry standard technology for pI determination and charge variant quantification of proteins and antibodies. However, the charge variants of PEGylated proteins merge into one broad peak during iCIEF, most likely due to masking of proteins by the surrounding PEG chain as well as the increased hydrodynamic volume due to PEGylation. Here, we report our novel matrix formula with a combination of glycine and taurine that significantly improved the separation of charge variants in PEGylated proteins. As a result, it is no longer necessary to conduct IEF of proteins prior to PEGylation, which does not reflect the changes caused by PEGylation and purification processes. The novel matrix (glycine and taurine) enables iCIEF analysis of PEGylated proteins in their real conjugated states.

Minimization of artifact protein aggregation using tetradecyl sulfate and hexadecyl sulfate in capillary gel electrophoresis under reducing conditions.

In the biopharmaceutical industry, CE-SDS assesses the purity, heterogeneity, and stability of therapeutic proteins. However, for mAb-1 and mAb-2, typical CE-SDS under reducing conditions produced atypical protein peak profiles, which led to biased purity results, thus were not acceptable for biologics manufacturing. This bias was caused by the formation of method-induced higher molecular weight artifacts, the levels of which correlated with protein concentration. Here we show that adding sodium tetradecyl and hexadecyl sulfates to the sample and the sieving gel buffer solutions was required to prevent formation of aggregate artifacts and to maintain detergent:protein uniformity, suggesting their importance during the sample preparation steps of heat denaturation and subsequent cooling as well as during capillary migration. For these proteins, we show that this uniformity was likely due to the ability of these detergents to bind proteins with markedly higher affinities compared to SDS. "CE-SCX S" methods (where CE-SCX S is CGE using detergent composed of a sodium sulfate head group and a hydrocarbon tail, with "CX " representing various tail lengths), were developed with a sodium tetradecyl sulfate sample buffer and a sodium hexadecyl sulfate containing sieving gel buffer that minimized artifacts and provided robust characterization and release results for mAb-1 and mAb-2.

Online/at-line measurement, analysis and control of product titer and critical product quality attributes (CQAs) during process development.

Process analytical technology (PAT) has been defined by the Food and Drug Administration as a system for designing, analyzing, and controlling manufacturing through timely measurements to ensure final product quality. Based on quality-by-design (QbD) principles, real-time or near-real-time data monitoring is essential for timely control of critical quality attributes (CQAs) to keep the process in a state of control. To facilitate next-generation continuous bioprocessing, deployment of PAT tools for real-time monitoring is integral for process understanding and control. Real-time monitoring and control of CQAs are essential to keep the process within the design space and align with the guiding principles of QbD. The contents of this manuscript are pertinent to the online/at-line monitoring of upstream titer and downstream product quality with timely process control. We demonstrated that an ultra-performance liquid chromatography (UPLC) system interfaced with a UPLC-process sample manager (UPLC-PSM) can be utilized to measure titer and CQAs directly from bioreactors and downstream unit operations, respectively. We established online titer measurements from fed-batch and perfusion-based alternating tangential flow bioreactors as well as product quality assessments of downstream operations for real-time peak collection. This integrated, fully automated system for online data monitoring with feedback control is designed to achieve desired product quality.

Technology outlook for real-time quality attribute and process parameter monitoring in biopharmaceutical development-A review.

Real-time monitoring of bioprocesses by the integration of analytics at critical unit operations is one of the paramount necessities for quality by design manufacturing and real-time release (RTR) of biopharmaceuticals. A well-defined process analytical technology (PAT) roadmap enables the monitoring of critical process parameters and quality attributes at appropriate unit operations to develop an analytical paradigm that is capable of providing real-time data. We believe a comprehensive PAT roadmap should entail not only integration of analytical tools into the bioprocess but also should address automated-data piping, analysis, aggregation, visualization, and smart utility of data for advanced-data analytics such as machine and deep learning for holistic process understanding. In this review, we discuss a broad spectrum of PAT technologies spanning from vibrational spectroscopy, multivariate data analysis, multiattribute chromatography, mass spectrometry, sensors, and automated-sampling technologies. We also provide insights, based on our experience in clinical and commercial manufacturing, into data automation, data visualization, and smart utility of data for advanced-analytics in PAT. This review is catered for a broad audience, including those new to the field to those well versed in applying these technologies. The article is also intended to give some insight into the strategies we have undertaken to implement PAT tools in biologics process development with the vision of realizing RTR testing in biomanufacturing and to meet regulatory expectations.

Real-time monitoring of quality attributes by in-line Fourier transform infrared spectroscopic sensors at ultrafiltration and diafiltration of bioprocess.

Technologies capable of monitoring product quality attributes and process parameters in real time are becoming popular due to the endorsement of regulatory agencies and also to support the agile development of biotherapeutic pipelines. The utility of vibrational spectroscopic techniques such as Fourier transform mid-infrared (Mid-IR) and multivariate data analysis (MVDA) models allows the prediction of multiple critical attributes simultaneously in real time. This study reports the use of Mid-IR and MVDA model sensors for monitoring of multiple attributes (excipients and protein concentrations) in real time (measurement frequency of every 40 s) at ultrafiltration and diafiltration (UF/DF) unit operation of biologics manufacturing. The platform features integration of fiber optic Mid-IR probe sensors to UF/DF set up at the bulk solution and through a flow cell at the retentate line followed by automated Mid-IR data piping into a process monitoring software platform with pre-loaded partial least square regression (PLS) chemometric models. Data visualization infrastructure is also built-in to the platform so that upon automated PLS prediction of excipients and protein concentrations, the results were projected in a graphical or numerical format in real time. The Mid-IR predicted concentrations of excipients and protein show excellent correlation with the offline measurements by traditional analytical methods. Absolute percent difference values between Mid-IR predicted results and offline reference assay results were ≤5% across all the excipients and the protein of interest; which shows a great promise as a reliable process analytical technology tool.

Assessment of CE-based baseline disturbances using simulation and targeted experimental evaluation-impact on the purity determination of therapeutic proteins.

The baseline instability for capillary electrophoretic analysis is an intrinsic feature of the technique, which has not been thoroughly examined for its impact on therapeutic protein purity analysis with the capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) applications. For the particular CE-SDS application, this phenomenon was manifested through peak migration time shifts and sliding of the superimposed baseline profile. These dual phenomena are closely associated so that experimental assessment alone may not shed enough light to the underlying drivers. In the current study, both experimental and simulation approaches were employed to assess the systematic drifts. Computer simulation was used to decipher the two underlying factors and test their contributions toward purity and impurity peak determination inaccuracies. The data generated in this study demonstrated that the electrophoretic baseline disturbance had more pronounced impact on the purity data than the migration time shift. In addition, the potential contributing factors to the baseline disturbances were assessed experimentally which indicated that the source is related to thermal disruption during a sample run and the unique baseline patterns came from the background electrolytes. To improve data reproducibility for drug purity testing in the industrial setting and quality control (QC) environment, it is recommended to run shorter injection sequences including fewer samples and closely monitor the baseline drift for accurate integration. Those methods would help reduce the impact of systematic drift and disturbances. Graphical abstract.

Therapeutic protein purity and fragmented species characterization by capillary electrophoresis sodium dodecyl sulfate using systematic hybrid cleavage and forced degradation.

Positive identification of capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) electropherogram peaks provides information to understand protein molecular characteristics at the structural level. It is critical in the design of a robust assay that can accurately resolve, differentiate, and quantify all therapeutic protein components including fragmented species, which are considered as product related impurities. However, direct identification of the impurity peaks observed in CE-SDS is a challenging and oftentimes an ambiguous task. This paper proposed a systematic workflow for characterizing CE-SDS fragmentation peaks. Forced degradation of monoclonal antibody (mAb) by multiple stress methods was utilized to induce fragmentation and species enrichment. The characteristics, such as size and the clipped region of sequence, were then evaluated based on multiple enzymatic treatment and particle reduction. The identified fragments were further confirmed using tryptic digestion and liquid chromatography coupled with mass spectrometry (LC-MS) analysis. Common fragment sizes and clipping locations are identified after evaluating multiple IgG molecules. The methodology and procedure described in this article are readily deployable and will provide necessary information for method, process, and product characterizations. Graphical abstract.

A Comprehensive Review of Analgesia and Pain Modalities in Hip Fracture Pathogenesis.

PURPOSE OF REVIEW: Hip fracture is common in the elderly population, painful and costly. The present investigation was undertaken to review epidemiology, socio-economic and medical implications, relevant anatomy, and anesthetic and pain modalities of hip fracture. RECENT FINDINGS: A literature search of PubMed, Ovid Medline, and Cochrane databases was conducted in December 2018 to identify relevant published clinical trials, review articles, and meta-analyses studies related to anesthetic and pain modalities of hip fracture. The acute pain management in these situations is often challenging. Common issues associated with morbidity and mortality include patients' physiological decrease in function, medical comorbidities, and cognitive impairment, which all can confound and complicate pain assessment and treatment. Perioperative multidisciplinary and multimodal approaches require medical, surgical, and anesthesiology teams employing adequate preoperative optimization. Reduction in pain and disability utilizing opioid and non-opioid therapies, regional anesthesia, patient-tailored anesthetic approach, and delirium prevention strategies seems to ensure best outcomes.

Dosimetric comparison of distal esophageal carcinoma plans for patients treated with small-spot intensity-modulated proton versus volumetric-modulated arc therapies.

BACKGROUND: Esophageal carcinoma is the eighth most common cancer in the world. Volumetric-modulated arc therapy (VMAT) is widely used to treat distal esophageal carcinoma due to high conformality to the target and good sparing of organs at risk (OAR). It is not clear if small-spot intensity-modulated proton therapy (IMPT) demonstrates a dosimetric advantage over VMAT. In this study, we compared dosimetric performance of VMAT and small-spot IMPT for distal esophageal carcinoma in terms of plan quality, plan robustness, and interplay effects. METHODS: 35 distal esophageal carcinoma patients were retrospectively reviewed; 19 patients received small-spot IMPT and the remaining 16 of them received VMAT. Both plans were generated by delivering prescription doses to clinical target volumes (CTVs) on phase-averaged 4D-CT's. The dose-volume-histogram (DVH) band method was used to quantify plan robustness. Software was developed to evaluate interplay effects with randomized starting phases for each field per fraction. DVH indices were compared using Wilcoxon rank-sum test. For fair comparison, all the treatment plans were normalized to have the same CTVhigh D95% in the nominal scenario relative to the prescription dose. RESULTS: In the nominal scenario, small-spot IMPT delivered statistically significantly lower liver Dmean and V30Gy[RBE] , lung Dmean , heart Dmean compared with VMAT. CTVhigh dose homogeneity and protection of other OARs were comparable between the two treatments. In terms of plan robustness, the IMPT and VMAT plans were comparable for kidney V18Gy[RBE] , liver V30Gy[RBE] , stomach V45Gy[RBE] , lung Dmean , V5Gy[RBE] , and V20Gy[RBE] , cord Dmax and D 0.03 c m 3 , liver Dmean , heart V20Gy[RBE] , and V30Gy[RBE] , but IMPT was significantly worse for CTVhigh D95% , D 2 c m 3 , and D5% -D95% , CTVlow D95% , heart Dmean , and V40Gy[RBE] , requiring careful and experienced adjustments during the planning process and robustness considerations. The small-spot IMPT plans still met the standard clinical requirements after interplay effects were considered. CONCLUSIONS: Small-spot IMPT decreases doses to heart, liver, and total lung compared to VMAT as well as achieves clinically acceptable plan robustness. Our study supports the use of small-spot IMPT for the treatment of distal esophageal carcinoma.

Imaged capillary isoelectric focusing in native condition: A novel and successful example.

Imaged capillary isoelectric focusing (icIEF) separates ampholytic components of biomolecules in an electric field according to their isoelectric points and has been used for protein charge variants quantification and characterization. Denaturants are ordinarily incorporated into icIEF to stabilize charge species in solution. In certain circumstances, however, denaturants are detrimental to stable isoelectric separation of proteins due to their unique structural and biophysical features, such as an aggregation-prone antibody we encountered recently. Here we report our novel matrix formula non-detergent sulfobetaine and taurine (NDSB-T). It is deprived of denaturants that notably ameliorates the assay robustness and peak resolution for this antibody. NDSB-T is a combination of non-detergent sulfobetaine (NDSB) and taurine possessing the stabilization and separation power while maintaining protein integrity. As a result, assay throughputs are tremendously increased for more than 10 folds along with extraordinarily improved assay accuracy. Furthermore, NDSB-T can separate and quantify protein charge species in native state and therefore avoid partial denaturation derived peaks which are often misleading and hard to characterize. NDSB-T may be a valuable tool for proteins incompatible with conventional icIEF matrices and potentially opens a new window for icIEF assay in native conditions.

Sex differences and similarities in the neuroendocrine regulation of social behavior in an African cichlid fish.

An individual's position in a social hierarchy profoundly affects behavior and physiology through interactions with community members, yet little is known about how the brain contributes to status differences between and within the social states or sexes. We aimed to determine sex-specific attributes of social status by comparing circulating sex steroid hormones and neural gene expression of sex steroid receptors in dominant and subordinate male and female Astatotilapia burtoni, a highly social African cichlid fish. We found that testosterone and 17ß-estradiol levels are higher in males regardless of status and dominant individuals regardless of sex. Progesterone was found to be higher in dominant individuals regardless of sex. Based on pharmacological manipulations in males and females, progesterone appears to be a common mechanism for promoting courtship in dominant individuals. We also examined expression of androgen receptors, estrogen receptor α, and the progesterone receptor in five brain regions that are important for social behavior. Most of the differences in brain sex steroid receptor expression were due to sex rather than status. Our results suggest that the parvocellular preoptic area is a core region for mediating sex differences through androgen and estrogen receptor expression, whereas the progesterone receptor may mediate sex and status behaviors in the putative homologs of the nucleus accumbens and ventromedial hypothalamus. Overall our results suggest sex differences and similarities in the regulation of social dominance by gonadal hormones and their receptors in the brain.