Biomedical Engineering

Rifamycins are a class of antibiotics that were first discovered in 1957 and are known for their use in treating tuberculosis (TB). Rifamycins exhibit bactericidal activity against many Gram-positive and Gram-negative bacteria by inhibiting RNA polymerase (RNAP); however, resistance is prevalent and the mechanisms range from primary target modification and antibiotic inactivation to cytoplasmic exclusion. Further, phenotypic resistance, in which only a subpopulation of bacteria grow in concentrations exceeding their minimum inhibitory concentration, and tolerance, which is characterized by reduced rates of bacterial cell death, have been identified as additional causes of rifamycin failure. Here we summarize current understanding and recent developments regarding this critical antibiotic class.

We tested the relation between pain behavior, theta (4–8 Hz) oscillations in somatosensory cortex and burst firing in thalamic neurons in vivo. Optically-induced thalamic bursts attenuated cortical theta and mechanical allodynia. It is proposed that thalamic bursts are an adaptive response to pain that de-synchronizes cortical theta and decreases sensory salience. Evidence for augmented theta (4–8 Hz) oscillations in primary somatosensory (SI) cortex was initially reported in chronic pain patients using electroencephalography (EEG) 1 , followed by data in animal pain models using EEG 2 , electrocorticography 3 and intracortical local field potential (LFP) recordings 4. Excess theta power is attenuated upon pain relief following thalamic lesion in humans 5 and analgesic drug treatments in animal models 2, 4. Though generators of theta oscillations remain unidentified, sensory thalamus is a likely contributor 4, 6 given its unique ability to gate sensory information and to modulate cortical oscillations required for optimal behavioral responses 7. Thalamocortical neurons fire in two dynamic and state dependent modes: tonic and high frequency or 'burst' discharges of two or more action potentials 8. The respective roles of these modes in gating sensory processing remains controversial 9, 10. Burst patterns have been characterized pre-clinically 11–13 and clinically 14, 15 during pain states. However, conflicting evidence suggests thalamic bursts may be positively 4, 6, 11–14 or negatively correlated with pain 16–20. Thalamic bursts are triggered predominantly by GABAergic drive from reticular thalamic nucleus (TRN), a thin layer overlaying thalamus that receives strong input from limbic cortical areas conveying information related to emotion and attention 21. Pharmacologic and molecular data further suggest that the GABA-mediated inhibitory tone in thalamus is suppressed during pain 22, 23 , presumably due to inhibition of TRN neurons 24, 25. We tested the hypothesis that rescuing TRN's 'gating' function by selective optical stimulation releases TRN neurons from inhibition, thus promoting thalamic bursting, reducing cortical theta and reversing nociceptive behavior. We reasoned that a multidisciplinary approach combining electrophysiology, optogenetics and behavior to probe this important but under-appreciated question in a longitudinal study design would provide the strongest evidence to date for a causal spatiotemporal relation between micro-scale unitary bursting in thalamus and macro-scale LFP oscillations in cortex. To selectively induce thalamic bursts, we optically stimulated TRN neurons in awake, unrestrained trans-genic mice (VGAT) co-expressing the vesicular GABA transporter with Channelrhodopsin-2 (ChR2). We used a custom-made multi-channel system 26 to record extracellularly from putative single-units in ventral poste-rolateral (VPL) thalamus and LFP in SI hindlimb area 26–29 (Fig. 1a,b). Histological analysis confirmed ChR2 expression limited to GABAergic neurons in TRN (Fig. 1c,d; additional criteria for tetrode localization shown in Supplemental Fig. 1). In a pilot experiment using naive VGAT mice, we confirmed that optical stimulation at 10 Hz (consistent with the physiological 'baseline' firing rate of TRN neurons 30) effectively reduced SI theta power in sedated mice (Fig. 2a; TRN stimulation at 0.5, 10 and 50 Hz reduced SI theta power to 5.14 × 10 −2 ± 0.71 × 10 −2 mV 2 , 4.35 × 10 −2 ± 0.33 × 10 −2 mV 2 and 4.45 × 10 −2 ± 0.36 × 10 −2 mV 2 respectively compared to baseline 5.48 × 10 −2 ± 0.38 × 10 −2 mV 2). We then confirmed in awake, resting mice that TRN

Ferroglobus placidus was discovered to oxidize completely the aromatic amino acids tyrosine, phenylalanine and tryptophan when Fe(III) oxide was provided as an electron acceptor. This property had not been reported previously for a hyperthermophilic archaeon. It appeared that F. placidus follows a pathway for phenylalanine and tryptophan degradation similar to that of mesophilic nitrate-reducing bacteria, Thauera aromatica and Aromatoleum aromaticum EbN1. Phenylacetate, 4-hydroxyphenylacetate and indole-3-acetate were formed during anaerobic degradation of phenylalanine, tyrosine and tryptophan, respectively. Candidate genes for enzymes involved in the anaerobic oxidation of phenylalanine to phenylacetate (phenylalanine transaminase, phenylpyruvate decarboxylase and phenylacetaldehyde : ferredoxin oxidoreductase) were identified in the F. placidus genome. In addition, transcription of candidate genes for the anaerobic phenylacetate degradation, benzoyl-CoA degradation and glutaryl-CoA degradation pathways was significantly upregulated in microarray and quantitative real-time-PCR studies comparing phenylacetate-grown cells with acetate-grown cells. These results suggested that the general strategies for anaerobic degradation of aromatic amino acids are highly conserved amongst bacteria and archaea living in both mesophilic and hyperthermophilic environments. They also provided insights into the diverse metabolism of Archaeoglobaceae species living in hyperthermophilic environments.

Emerging cell therapies have created new demands for instruments that will increase processing efficiency. Purification of lymphocytes prior to downstream steps of gene transfer currently relies on centrifugal separation, which has drawbacks in output sample purity and process automation. Here, we present an alternative approach to blood cell purification using acoustic forces in plastic microchannels. We provide details regarding the system's ability to purify lymphocytes relative to other blood cell types while maintaining a high overall recovery, testing performance starting from leukapheresis product, buffy coat, and whole blood. Depending on settings, the device achieves for lymphocytes up to 97% purity and up to 68% recovery, and depletes 98% of monocytes while also reducing red cells and platelets. We expect that future scale-up of our system for increased throughput will enable its incorporation in the cell therapy workflow, and that it could ultimately reduce costs and expand access for patients.

Substitutions that disrupt pre-mRNA splicing are a common cause of genetic disease. On average, 13.4% of all hereditary disease alleles are classified as splicing mutations mapping to the canonical 5' and 3' splice sites. However, splicing mutations present in exons and deeper intronic positions are vastly underreported. A recent re-analysis of coding mutations in exon 10 of the Lynch Syndrome gene, MLH1, revealed an extremely high rate (77%) of mutations that lead to defective splicing. This finding is confirmed by extending the sampling to five other exons in the MLH1 gene. Further analysis suggests a more general phenomenon of defective splicing driving Lynch Syndrome. Of the 36 mutations tested, 11 disrupted splicing. Furthermore, analyzing past reports suggest that MLH1 mutations in canonical splice sites also occupy a much higher fraction (36%) of total mutations than expected. When performing a comprehensive analysis of splicing mutations in human disease genes, we fo...

Humans co-existed and interbred with other hominins which later became extinct. These archaic hominins are known to us only through fossil records and for two cases, genome sequences. Here we engineer Neanderthal and Denisovan sequences into thousands of artificial genes to reconstruct the pre-mRNA processing patterns of these extinct populations. Of the 5,224 alleles tested in this massively parallel splicing reporter assay (MaPSy), we report 969 exonic splicing mutations (ESMs) that correspond to differences in exon recognition between extant and extinct hominins. Using MaPSy splicing variants, predicted splicing variants, and splicing quantitative trait loci, we show that splice-disrupting variants experienced greater purifying selection in anatomically modern humans than in Neanderthals. Adaptively introgressed variants were enriched for moderate effect splicing variants, consistent with positive selection for alternative spliced alleles following introgression. As particularly ...

Over 400,000 Americans are intubated in emergency settings annually, with indications ranging from respiratory failure to airway obstructions to anaphylaxis. About 12.7% of emergency intubations are unsuccessful on the first attempt. Failure to intubate on the first attempt is associated with a higher likelihood of adverse events, including oxygen desaturation, aspiration, trauma to soft tissue, dysrhythmia, hypotension, and cardiac arrest. Difficult airways, as classified on an established clinical scale, are found in up to 30% of emergency department (ED) patients and are a significant contributor to failure to intubate. Difficult intubations have been associated with longer lengths of stay and significantly greater costs than standard intubations. There exists a wide range of airway management devices, both invasive and noninvasive, which are available in the emergency setting to accommodate difficult airways. Yet, first-pass success rates remain variable and leave room for improvement. In this article, we review the disease states most correlated with intubation, the current landscape of emergency airway management technologies, and the market potential for innovation. The aim of this review is to inspire new technologies to assist difficult airway management, given the substantial opportunity for translation due to two key-value signposts of medical innovation: the potential to decrease cost and the potential to improve clinical outcomes.

To determine the contribution of defective splicing in Autism Spectrum Disorders (ASD), the most common neurodevelopmental disorder, a high throughput Massively Parallel Splicing Assay (MaPSY) was employed and identified 42 exonic splicing mutants out of 725 coding de novo variants discovered in the sequencing of ASD families. A redesign of the minigene constructs in MaPSY revealed that upstream exons with strong 5' splice sites increase the magnitude of skipping phenotypes observed in downstream exons. Select hits were validated by RT-PCR and amplicon sequencing in patient cell lines. Exonic splicing mutants were enriched in probands relative to unaffected siblings-especially synonymous variants (7.5% vs 3.5%, respectively). Of the 26 genes disrupted by exonic splicing mutations, 6 were in known ASD genes and 3 were in paralogs of known ASD genes. Of particular interest was a synonymous variant in TNRC6C-an ASD gene paralog with interactions with other ASD genes. Clinical records of 3 ASD patients with TNRC6C variant revealed respiratory issues consistent with phenotypes observed in TNRC6 depleted mice. Overall, this study highlights the need for splicing analysis in determining variant pathogenicity, especially as it relates to ASD.

Over 400,000 Americans are intubated in emergency settings annually, with indications ranging from respiratory failure to airway obstructions to anaphylaxis. About 12.7% of emergency intubations are unsuccessful on the first attempt. Failure to intubate on the first attempt is associated with a higher likelihood of adverse events, including oxygen desaturation, aspiration, trauma to soft tissue, dysrhythmia, hypotension, and cardiac arrest. Difficult airways, as classified on an established clinical scale, are found in up to 30% of emergency department (ED) patients and are a significant contributor to failure to intubate. Difficult intubations have been associated with longer lengths of stay and significantly greater costs than standard intubations. There exists a wide range of airway management devices, both invasive and noninvasive, which are available in the emergency setting to accommodate difficult airways. Yet, first-pass success rates remain variable and leave room for improvement. In this article, we review the disease states most correlated with intubation, the current landscape of emergency airway management technologies, and the market potential for innovation. The aim of this review is to inspire new technologies to assist difficult airway management, given the substantial opportunity for translation due to two key-value signposts of medical innovation: the potential to decrease cost and the potential to improve clinical outcomes.

Substitutions that disrupt pre-mRNA splicing are a common cause of genetic disease. On average, 13.4% of all hereditary disease alleles are classified as splicing mutations mapping to the canonical 5' and 3' splice sites. However, splicing mutations present in exons and deeper intronic positions are vastly underreported. A recent re-analysis of coding mutations in exon 10 of the Lynch Syndrome gene, MLH1, revealed an extremely high rate (77%) of mutations that lead to defective splicing. This finding is confirmed by extending the sampling to five other exons in the MLH1 gene. Further analysis suggests a more general phenomenon of defective splicing driving Lynch Syndrome. Of the 36 mutations tested, 11 disrupted splicing. Furthermore, analyzing past reports suggest that MLH1 mutations in canonical splice sites also occupy a much higher fraction (36%) of total mutations than expected. When performing a comprehensive analysis of splicing mutations in human disease genes, we fo...

Humans co-existed and interbred with other hominins which later became extinct. These archaic hominins are known to us only through fossil records and for two cases, genome sequences. Here we engineer Neanderthal and Denisovan sequences into thousands of artificial genes to reconstruct the pre-mRNA processing patterns of these extinct populations. Of the 5,224 alleles tested in this massively parallel splicing reporter assay (MaPSy), we report 969 exonic splicing mutations (ESMs) that correspond to differences in exon recognition between extant and extinct hominins. Using MaPSy splicing variants, predicted splicing variants, and splicing quantitative trait loci, we show that splice-disrupting variants experienced greater purifying selection in anatomically modern humans than in Neanderthals. Adaptively introgressed variants were enriched for moderate effect splicing variants, consistent with positive selection for alternative spliced alleles following introgression. As particularly ...