HIGH THROUGHPUT QUANTITATION OF HUMAN NEUTROPHIL RECRUITMENT AND FUNCTIONAL RESPONSES IN AN AIR-BLOOD BARRIER ARRAY.

Dysregulated neutrophil recruitment drives many pulmonary diseases, but most preclinical screening methods are unsuited to evaluate pulmonary neutrophilia, limiting progress towards therapeutics. Namely, high throughput therapeutic screening systems typically exclude critical neutrophilic pathophysiology, including blood-to-lung recruitment, dysfunctional activation, and resulting impacts on the air-blood barrier. To meet the conflicting demands of physiological complexity and high throughput, we developed an assay of 96-well Leukocyte recruitment in an Air-Blood Barrier Array (L-ABBA-96) that enables in vivo -like neutrophil recruitment compatible with downstream phenotyping by automated flow cytometry. We modeled acute respiratory distress syndrome (ARDS) with neutrophil recruitment to 20 ng/mL epithelial-side interleukin 8 (IL-8) and found a dose dependent reduction in recruitment with physiologic doses of baricitinib, a JAK1/2 inhibitor recently FDA-approved for severe COVID-19 ARDS. Additionally, neutrophil recruitment to patient-derived cystic fibrosis sputum supernatant induced disease-mimetic recruitment and activation of healthy donor neutrophils and upregulated endothelial e-selectin. Compared to 24-well assays, the L-ABBA-96 reduces required patient sample volumes by 25 times per well and quadruples throughput per plate. Compared to microfluidic assays, the L-ABBA-96 recruits two orders of magnitude more neutrophils per well, enabling downstream flow cytometry and other standard biochemical assays. This novel pairing of high-throughput in vitro modeling of organ-level lung function with parallel high-throughput leukocyte phenotyping substantially advances opportunities for pathophysiological studies, personalized medicine, and drug testing applications.

Cationic cholesterol-dependent LNP delivery to lung stem cells, the liver, and heart.

Adding a cationic helper lipid to a lipid nanoparticle (LNP) can increase lung delivery and decrease liver delivery. However, it remains unclear whether charge-dependent tropism is universal or, alternatively, whether it depends on the component that is charged. Here, we report evidence that cationic cholesterol-dependent tropism can differ from cationic helper lipid-dependent tropism. By testing how 196 LNPs delivered mRNA to 22 cell types, we found that charged cholesterols led to a different lung:liver delivery ratio than charged helper lipids. We also found that combining cationic cholesterol with a cationic helper lipid led to mRNA delivery in the heart as well as several lung cell types, including stem cell-like populations. These data highlight the utility of exploring charge-dependent LNP tropism.

Liquid plug propagation in computer-controlled microfluidic airway-on-a-chip with semi-circular microchannels.

This paper introduces a two-inlet, one-outlet lung-on-a-chip device with semi-circular cross-section microchannels and computer-controlled fluidic switching that enables a broader systematic investigation of liquid plug dynamics in a manner relevant to the distal airways. A leak-proof bonding protocol for micro-milled devices facilitates channel bonding and culture of confluent primary small airway epithelial cells. Production of liquid plugs with computer-controlled inlet channel valving and just one outlet allows more stable long-term plug generation and propagation compared to previous designs. The system also captures both plug speed and length as well as pressure drop concurrently. In one demonstration, the system reproducibly generates surfactant-containing liquid plugs, a challenging process due to lower surface tension that makes the plug formation less stable. The addition of surfactant decreases the pressure required to initiate plug propagation, a potentially significant effect in diseases where surfactant in the airways is absent or dysfunctional. Next, the device recapitulates the effect of increasing fluid viscosity, a challenging analysis due to higher resistance of viscous fluids that makes plug formation and propagation more difficult particularly in airway-relevant length scales. Experimental results show that increased fluid viscosity decreases plug propagation speed for a given air flow rate. These findings are supplemented by computational modeling of viscous plug propagation that demonstrates increased plug propagation time, increased maximum wall shear stress, and greater pressure differentials in more viscous conditions of plug propagation. These results match physiology as mucus viscosity is increased in various obstructive lung diseases where it is known that respiratory mechanics can be compromised due to mucus plugging of the distal airways. Finally, experiments evaluate the effect of channel geometry on primary human small airway epithelial cell injury in this lung-on-a-chip. There is more injury in the middle of the channel relative to the edges highlighting the role of channel shape, a physiologically relevant parameter as airway cross-sectional geometry can also be non-circular. In sum, this paper describes a system that pushes the device limits with regards to the types of liquid plugs that can be stably generated for studies of distal airway fluid mechanical injury.

Rational design of the genetic code expansion toolkit for in vivo encoding of D-amino acids.

Once thought to be non-naturally occurring, D-amino acids (DAAs) have in recent years been revealed to play a wide range of physiological roles across the tree of life, including in human systems. Synthetic biologists have since exploited DAAs' unique biophysical properties to generate peptides and proteins with novel or enhanced functions. However, while peptides and small proteins containing DAAs can be efficiently prepared in vitro, producing large-sized heterochiral proteins poses as a major challenge mainly due to absence of pre-existing DAA translational machinery and presence of endogenous chiral discriminators. Based on our previous work demonstrating pyrrolysyl-tRNA synthetase's (PylRS') remarkable substrate polyspecificity, this work attempts to increase PylRS' ability in directly charging tRNAPyl with D-phenylalanine analogs (DFAs). We here report a novel, polyspecific Methanosarcina mazei PylRS mutant, DFRS2, capable of incorporating DFAs into proteins via ribosomal synthesis in vivo. To validate its utility, in vivo translational DAA substitution were performed in superfolder green fluorescent protein and human heavy chain ferritin, successfully altering both proteins' physiochemical properties. Furthermore, aminoacylation kinetic assays further demonstrated aminoacylation of DFAs by DFRS2 in vitro.

Serosurveillance of SARS-CoV-2 in companion animals in Sarawak, Malaysia.

SARS-CoV-2 is a zoonotic betacoronavirus that was first reported at the dawn of 2019 in Wuhan, China and has since spread globally, causing an ongoing pandemic. Anthroponotic transmission was reported early, with confirmed infections reported in 26 species to date, including dogs and cats. However, there is a paucity of reports on the transmission of SARS-CoV-2 to companion animals, and thus, we aimed to estimate the seroprevalence of SARS-CoV-2 in dogs and cats in Sarawak, Malaysia. From August 2022 to 2023, we screened plasma samples of 172 companion animals in Sarawak, Malaysia, using a species-independent surrogate virus neutralization test. Our findings revealed the presence of neutralizing antibodies of SARS-CoV-2 in 24.5% (27/110) of dogs and 24.2% (15/62) of cats. To the best of our knowledge, this is the first report of the seroprevalence of SARS-CoV-2 in companion animals in Malaysia. Our findings emphasize the need for pet owners to distance themselves from their pets when unwell, and a strategy must be in place to monitor SARS-CoV-2 in companion animals to assess the potential impact of the virus on companion animals.

Liquid plug propagation in computer-controlled microfluidic airway-on-a-chip with semi-circular microchannels.

This paper introduces a two-inlet, one-outlet lung-on-a-chip device with semi-circular cross-section microchannels and computer-controlled fluidic switching that enables a broader systematic investigation of liquid plug dynamics in a manner relevant to the distal airways. A leak-proof bonding protocol for micro-milled devices facilitates channel bonding and culture of confluent primary small airway epithelial cells. Production of liquid plugs with computer-controlled inlet channel valving and just one outlet allows more stable long-term plug generation and propagation compared to previous designs. The system also captures both plug speed and length as well as pressure drop concurrently. In one demonstration, the system reproducibly generates surfactant-containing liquid plugs, a challenging process due to lower surface tension that makes the plug formation less stable. The addition of surfactant decreases the pressure required to initiate plug propagation, a potentially significant effect in diseases where surfactant in the airways is absent or dysfunctional. Next, the device recapitulates the effect of increasing fluid viscosity, a challenging analysis due to higher resistance of viscous fluids that makes plug formation and propagation more difficult particularly in airway-relevant length scales. Experimental results show that increased fluid viscosity decreases plug propagation speed for a given air flow rate. These findings are supplemented by computational modeling of viscous plug propagation that demonstrate increased plug propagation time, increased maximum wall shear stress, and greater pressure differentials in more viscous conditions of plug propagation. These results match physiology as mucus viscosity is increased in various obstructive lung diseases where it is known that respiratory mechanics can be compromised due to mucus plugging of the distal airways. Finally, experiments evaluate the effect of channel geometry on primary human small airway epithelial cell injury in this lung-on-a-chip. There is more injury in the middle of the channel relative to the edges highlighting the role of channel shape, a physiologically relevant parameter as airway cross-sectional geometry can also be non-circular. In sum, this paper describes a system that pushes the device limits with regards to the types of liquid plugs that can be stably generated for studies of distal airway fluid mechanical injury.

Regulations of myo-inositol homeostasis: Mechanisms, implications, and perspectives.

Phosphorylation is the most common module of cellular signalling pathways. The dynamic nature of phosphorylation, which is conferred by the balancing acts of kinases and phosphatases, allows this modification to finely control crucial cellular events such as growth, differentiation, and cell cycle progression. Although most research to date has focussed on protein phosphorylation, non-protein phosphorylation substrates also play vital roles in signal transduction. The most well-established substrate of non-protein phosphorylation is inositol, whose phosphorylation generates many important signalling molecules such as the second messenger IP3, a key factor in calcium signalling. A fundamental question to our understanding of inositol phosphorylation is how the levels of cellular inositol are controlled. While the availability of protein phosphorylation substrates is known to be readily controlled at the levels of transcription, translation, and/or protein degradation, the regulatory mechanisms that control the uptake, synthesis, and removal of inositol are underexplored. Potentially, such mechanisms serve as an important layer of regulation of cellular signal transduction pathways. There are two ways in which mammalian cells acquire inositol. The historic use of radioactive 3H-myo-inositol revealed that inositol is promptly imported from the extracellular environment by three specific symporters SMIT1/2, and HMIT, coupling sodium or proton entry, respectively. Inositol can also be synthesized de novo from glucose-6P, thanks to the enzymatic activity of ISYNA1. Intriguingly, emerging evidence suggests that in mammalian cells, de novo myo-inositol synthesis occurs irrespective of inositol availability in the environment, prompting the question of whether the two sources of inositol go through independent metabolic pathways, thus serving distinct functions. Furthermore, the metabolic stability of myo-inositol, coupled with the uptake and endogenous synthesis, determines that there must be exit pathways to remove this extraordinary sugar from the cells to maintain its homeostasis. This essay aims to review our current knowledge of myo-inositol homeostatic metabolism, since they are critical to the signalling events played by its phosphorylated forms.

Boredom and meaningful activity in adults experiencing homelessness: A mixed-methods study.

BACKGROUND.: Boredom has been reported as a frequent problem experienced by homeless persons, with implications for mental and social well-being. PURPOSE.: This study aimed to explore the nature and impact of boredom in the lives of homeless and formerly homeless persons. METHOD.: A mixed-methods design was used to engage 13 participants in a structured 92-item quantitative interview using six standardized measures, followed by a semistructured qualitative interview. Correlational analyses were performed with the data compiled from the quantitative interviews, and grounded theory strategies were used to analyze our qualitative data. The two analyses were integrated at the stage of interpretation. FINDINGS.: Boredom was described as a profound and pervasive experience for homeless persons, imposing deleterious impacts on mental well-being and driving substance use. A strong positive correlation between meaningful activity and mental well-being (r s = .767, p <. 01) and a strong negative correlation between boredom and belonging in one's community (r s = -.771, p < .01) were identified. IMPLICATIONS.: Boredom is a critical topic of study for occupational therapy in the area of homelessness. Future research is needed to design and test interventions to optimize the mental well-being, participation, and social connectedness of this population.

Dominance of orientation over frequency in the perception of 3-D slant and shape.

In images of textured three-dimensional surfaces, pattern changes can be characterized as changes in orientation and spatial frequency, features for which neurons in primary visual cortex are classically selective. Previously, we have demonstrated that correct 3-D shape perception is contingent on the visibility of orientation flows that run parallel to the surface curvature. We sought to determine the relative contributions of orientation modulations (OMs) and frequency modulations (FMs) for the detection of slant and shape from 3-D surfaces. Results show that 1) when OM and FM indicate inconsistent degrees of surface slant or curvature, observer responses were consistent with the slant or curvature specified by OM even if the FM indicated a slant or curvature in the opposite direction to the same degree. 2) For slanted surfaces, OM information dictates slant perception at both shallow and steep slants while FM information is effective only for steep slants. Together these results point to a dominant role of OM information in the perception of 3-D slant and shape.

Effects of texture component orientation on orientation flow visibility for 3-D shape perception.

In images of textured 3-D surfaces, orientation flows created by the texture components parallel to the surface slant play a critical role in conveying the surface slant and shape. This study examines the visibility of these orientation flows in complex patterns. Specifically, we examine the effect of orientation of neighboring texture components on orientation flow visibility. Complex plaids consisting of gratings equally spaced in orientation were mapped onto planar and curved surfaces. The visibility of the component that creates the orientation flows was quantified by measuring its contrast threshold (CT) while varying the combination of neighboring components present in the pattern. CTs were consistently lowest only when components closest in orientation to that of the orientation flows were subtracted from the pattern. This finding suggests that a previously reported frequency-selective cross-orientation suppression mechanism involved with the perception of 3-D shape from texture is affected by proximity in orientation of concurrent texture components.

Inconsistent condom use among young men who have sex with men, male sex workers, and transgenders in Thailand.

Young men who have sex with men (MSM) are at risk for HIV infection. We investigated inconsistent condom use among 827 sexually active young MSM (15-24 years), enrolled using venue-day-time sampling in Bangkok, Chiang Mai and Phuket, Thailand. Data was collected using palmtop computer-assisted self-interviewing. Of participants, 33.1% were regular MSM, 37.7% were male sex workers (MSWs) and 29.1% were transgenders (TGs). Of MSM, 46.7%, of MSWs, 34.9% and of TGs, 52.3% reported recent inconsistent condom use. In multivariate analysis, receptive anal intercourse (MSM, MSWs), receptive and insertive anal intercourse, living alone and a history of sexual coercion (MSWs), not carrying a condom when interviewed (MSM, TGs), lower education, worrying about HIV infection and a history of sexually transmitted infections (TGs) were significantly and independently associated with inconsistent condom use. Interventions for young MSM are needed and must consider the distinct risk factors of MSM, MSWs, and TGs.

Antiretroviral treatment outcome following genotyping in Thai children who failed dual nucleoside reverse transcriptase inhibitors.

OBJECTIVE: To evaluate outcomes in dual nucleoside reverse transcriptase inhibitor (NRTI) pretreated children after genotyping (GT). METHODS: We assessed CD4 and viral load (VL) in children three years after baseline GT at the time of dual NRTI failure. Baseline high grade resistance (HR) was defined as >or=4 nucleoside analogue mutations (NAMs)+/-Q151M or 69 insertion complex, and low grade resistance (LR) was defined as <4 NAMs. Genotypic susceptibility scores (GSS) were determined. The current selection of antiretrovirals (ARV) was based on physician judgment and ARV availability. RESULTS: Seventy-two children were enrolled, with a mean age of 9.3 years; 61% were female. Baseline median CD4 was 18%, VL was 1.7 log(10) with HR 37.5%, LR 56.9% and no mutation (NR, no resistance) 5.6%. Sixty-five (90.3%) switched ARV: 46.2% non-nucleoside reverse transcriptase inhibitor (NNRTI), 30.8% protease inhibitor (PI), and 23.1% PI+NNRTI based highly active antiretroviral therapy (HAART). The choice of regimen did not differ based on baseline HR, LR, and NR. The median duration from dual NRTI therapy to HAART was 5.4 years (interquartile range (IQR) 4.0-6.9 years) and the mean (SD) duration of current HAART regimen was 1.51 (1.78) years; both were similar between ARV groups. Five children continued dual NRTI, two interrupted therapy. The GSS score was significantly higher in the PI group (3.1) vs. PI+NNRTI (2.5) vs. NNRTI (2.6) groups. Sixty-three percent of the HR group used PI or PI+NNRTI-based HAART compared to 41% of the LR group, p=not significant. At follow-up, median CD4 changes from baseline were +5% and VL -2.2 log(10) (p<0.001). VL <1.7 log(10) was seen in 59.3% of HR, 58.5% of LR, and 50.0% of NR groups (no significant difference). More children on PI (75%) and PI+NNRTI (80%) based HAART had VL <50 compared to NNRTI-based HAART (50%), p=0.003. CONCLUSION: PI-based regimens showed a higher rate of undetectable VL compared with NNRTI-based regimens. Having GT may not affect second-line treatment choices in developing countries, most likely due to late VL failure and limited availability of PIs.

Release from cross-orientation suppression facilitates 3D shape perception.

Cross-orientation suppression (COS) in striate cortex has been implicated in the efficient encoding of visual stimuli. We show that release from COS facilitates the decoding of 3-D shape. In planar surfaces overlaid with textures, slanting the surface can increase the visibility of the component parallel to the slant. Since this component provides the orientation flows that signify 3-D shape, the enhancement of visibility facilitates 3-D slant perception. Contrast thresholds reveal that this enhancement results from a decrease in COS when 3-D slant creates a frequency mismatch between texture components. We show that coupling compressive nonlinearities in LGN neurons with expansive nonlinearities in cortical neurons can model the frequency-specific component of suppression.

Three-dimensional shape from second-order orientation flows.

In images of textured surfaces, orientation flows formed by perspective convergence invariably convey 3D shape. We show that orientation flows formed by contrast-modulated (CM) and illusory contours (IC) convey 3D shape, and that both stimulus types induce 3D shape aftereffects on CM and IC test stimuli. Adaptation to luminance-modulated (LM) orientation flows induce robust 3D shape aftereffects on CM and IC tests, however, aftereffects using CM/IC adapting stimuli on LM tests were substantially weaker. These results can be explained by the adaptation of 3D shape-selective neurons that invariantly extract first- and second-order orientation flows from striate and extra-striate signals, which receive stronger input from neurons selective for first-order orientation flows.

Sexual behavior and risk factors for HIV infection among homosexual and bisexual men in Thailand.

HIV prevalence and associated risk behaviors were examined among Thai bisexually active men (MSMW, n = 450) and men who have sex with men only (MSM-only, n = 1,125). Cross sectional venue-day-time sampling was used to collect data. Chi-square and logistic regression were used to identify HIV risk factors. HIV prevalence was 8.2% among MSMW and 21.2% among MSM-only. Consistent condom use with male partners was higher among MSMW (77.6%) than MSM-only (62.9%), and lower with female partners (44.4%). Lack of family confidant, migration, concern about acquiring HIV infection, and self-reported STD were associated with HIV prevalence among MSMW. Older age, lower educational level, residing in Bangkok or Chiang Mai, living away from family, recruitment from a sauna, increased frequency of visiting the surveyed venue, practicing receptive or both receptive and insertive anal intercourse, inconsistent condom use with male paying partners, and a history of drug use were associated with HIV prevalence in MSM-only.

Neural basis of 3-D shape aftereffects.

We used selective adaptation to identify the neural mechanisms responsible for 3-D shape perception from orientation flows in retinal images [Li, A., & Zaidi, Q. (2000). Perception of three-dimensional shape from texture is based on patterns of oriented energy. Vision Research 40 (2), 217-242)]. Three-dimensional shape adaptation could involve stages from photoreceptors to non-oriented retinal cells, oriented cells in striate cortex, and extra-striate cells that respond to 3-D slants. To psychophysically isolate the relevant stage, we used 3-D adapting stimuli created from real and illusory orientations, and test stimuli different from the adapting stimuli in phases and frequencies. The results showed that mechanisms that adapt to 3-D shapes combine real and illusory 2-D orientation information over a range of spatial frequencies.

Experiencing visuo-motor plasticity by prism adaptation in a classroom setting.

Neural plasticity is a key topic in the study of behavioral neuroscience, yet it can be a difficult concept to demonstrate in a classroom setting. In this report, we describe an interactive technique that can be used to demonstrate and quantify in a laboratory setting the plasticity of motor coordination to altered visual input, i.e. visuo-motor plasticity. Visual input can be easily altered by horizontally-displacing prism goggles. Open-loop motor coordination immediately after putting on these goggles is inaccurate. However, after performing a number of visuo-motor tasks wearing these goggles, coordination adapts and improves. Immediately after removing the goggles, a robust negative aftereffect resulting from adaptation to the goggles is consistently demonstrated. This negative aftereffect can be used to quantify the amount of adaptation that has taken place. We document how to create the prism goggles, how to quantify accuracy of motor coordination, what kinds of visuo-motor tasks consistently lead to significant adaptation, and the importance of active over passive adaptation conditions.

Three-dimensional shape perception from chromatic orientation flows.

The role of chromatic information in 3-D shape perception is controversial. We resolve this controversy by showing that chromatic orientation flows are sufficient for accurate perception of 3-D shape. Chromatic flows required less cone contrast to convey shape than did achromatic flows, thus ruling out luminance artifacts as a problem. Luminance artifacts were also ruled out by a protanope's inability to see 3-D shape from chromatic flows. Since chromatic orientation flows can only be extracted from retinal images by neurons that are responsive to color modulations and selective for orientation, the psychophysical results also resolve the controversy over the existence of such neurons. In addition, we show that identification of 3-D shapes from chromatic flows can be masked by luminance modulations, indicating that it is subserved by orientation-tuned neurons sensitive to both chromatic and luminance modulations.

Three-dimensional shape from non-homogeneous textures: carved and stretched surfaces.

We examined the perception of 3D shape for surfaces folded, carved, or stretched out of textured materials. The textures were composed of sums of sinusoidal gratings or of circular dots, and were designed to differentiate between orientation and frequency information present in perspective images of the surfaces. Correct perception of concavities, convexities, saddles, and slants required the visibility of signature patterns of orientation modulations. These patterns were identical to those identified previously for developable surfaces (A. Li & Q. Zaidi, 2000; Q. Zaidi & L. Li, 2002), despite the fact that textures were statistically homogeneous on developable surfaces but not on carved or stretched surfaces. Frequency modulations in the image were interpreted as cues to distance from the observer, which led to weak but qualitatively correct percepts for some carved and stretched surfaces but to misperceptions for others, similar to the misperceptions for developable surfaces (A. Li & Q. Zaidi, 2003). Irrespective of whether texture on the surface is homogeneous or non-homogeneous, similar neural modules can be used to locate signature orientation modulations and thus extract shape from texture cues.