Singapore-MIT Alliance for Research and Technology

Background The zonal organization of articular cartilage is critical for the biphasic mechanical properties of the tissue. Current treatments for articular cartilage have yet to regenerate this zonal architecture, compromising the functional efficacy of the repaired tissue, which could account for tissue failure in the long term. Autologous chondrocyte implantation (ACI) still suffers from inconsistent efficacy and a long recovery period stemming from implantation of a heterogeneous chondrocyte mixture. Hypothesis Stratified implantation of zonal chondrocytes would facilitate the recapitulation of articular cartilage zonal properties and improve the repair efficacy of ACI treatment. Study Design Controlled laboratory study. Methods Autologous chondrocytes extracted from porcine articular cartilage were subjected to dynamic microcarrier expansion followed by size-based segregation using a spiral microfluidic device for the enrichment of zonal chondrocytes. Zonal chondrocytes were implanted into a chondral defect as a bilayered hydrogel construct consisting of superficial zone chondrocytes overlaying middle/deep zone chondrocytes (n = 6). Twelve months after implantation, the repair efficacy was compared against implantation of full-thickness cartilage-derived heterogeneous chondrocytes expanded on tissue culture plates (n = 5) or microcarriers (n = 6). Results Quantitative assessment of the repair tissues, including gross morphology, histological analysis, micro–computed tomography (micro - CT), compression modulus, and surface lubrication analysis, at 12 months demonstrated statistically significant improvement in cartilage and subchondral bone repair with zonal chondrocyte bilayered implantation. Magnetic resonance imaging (MRI) T2 mapping indicated progressive improvement in graft maturation as early as 3 months, reaching normalcy at 9 months. Conclusion This study demonstrates that with appropriate expansion and isolation of zonal chondrocytes, stratified zonal chondrocyte implantation is able to facilitate restoration of articular cartilage zonal architecture and significantly enhance the functional repair as compared with current ACI treatment. Clinical Relevance With appropriate expansion and enrichment of zonal chondrocytes, stratified zonal chondrocyte implantation could represent a significant advancement over current ACI-based cartilage repair, with the potential to support quicker and better recovery.

Background Osteoarthritis (OA) is one of the most prevalent degenerative joint diseases, while the mechanism by which extracellular vesicles (EVs) promote chondrocyte regeneration remains unclear. The study assessed the effect of hypoxic mesenchymal stem cells (MSCs)-derived EVs on cartilage repair in a rat OA model. Methods The effects of EVs on chondrocyte regeneration and autophagy were evaluated in vitro. The influence of specific micro RNA (miRNA) and downstream target genes was examined following EV miRNA sequencing and multiple intersecting database analysis. Results We found EVs derived from hypoxia preconditioned human MSCs to promote cartilage repair in rat OA and enhance the proliferation and migration of chondrocytes in vitro, mediated via chondrocyte autophagy. MiRNA sequencing revealed a significant enrichment of miRNA122-5p in hypoxic MSCs EV, which through regulation of the target gene, DUSP2, mediated autophagy and participated in chondrocyte regeneration. DUSP2 regulation of chondrocyte autophagy could act via the phosphorylation of ERK1/2 and P38. Conclusions This study demonstrates that EVs released by MSCs under hypoxic conditions have a beneficial effect on chondrocyte regeneration. A novel mechanism for chondrocyte autophagy is mediated by miR122-5P and DUSP2 target molecules, providing new insights into OA treatments.

The first total synthesis of phenylpropanoid glycoside (PhG) isoacteoside 1 is presented. The synthesis employs a novel protection‐free strategy leveraging phenylboronic acid as a transient masking agent to enable regioselective mono‐rhamnosylation and caffeoylation. This method circumvents the need for traditional multi‐step protection and deprotection steps and enhances overall efficiency and yield. Key steps include a one‐pot, three‐step glycosylation yielding β‐D‐glucopyranoside, regioselective rhamnosylation via in‐situ boronate ester formation, selective caffeoylation at the OH‐6 position, and finally one‐pot deprotection to furnish isoacteoside in 26% overall yield. This work represents a significant advancement and serves as a model for preparing many PhGs with a sugar residue at OH‐2 and an acyl residue at OH‐6 of the 2‐phenylethyl‐β‐D‐glucoside core. This work sets a viable route for efficient PhG synthesis.

Chimeric Antigen Receptor (CAR) T cell therapy is a pivotal treatment for hematological malignancies. However, CAR T cell products exhibit batch-to-batch variability in cell number, quality, and in vivo efficacy due to donor-to-donor heterogeneity, and pre/post-manufacturing processes, and the manufacturing of such products necessitates careful testing, both post-manufacturing and pre-infusion. Here, we introduce the Cell Trajectory Modulation (CTM) assay, a microfluidic, label-free approach for the rapid evaluation of the functional attributes of CAR T cells based on biophysical features (i.e., size, deformability). CTM assay correlates with phenotypic metrics, including CD4:CD8 ratio, memory subtypes, and cytotoxic activity. Validated across multiple donors and culture platforms, the CTM assay requires fewer than 10,000 cells and delivers results within 10 minutes. Compared to labeled flow cytometry processing, the CTM assay offers real-time data to guide adaptive manufacturing workflows. Thus, the CTM assay offers an improvement over existing phenotypic assessments, marking a step forward in advancing CAR T cell therapy manufacturing.

Tropical peatlands are important global carbon sinks, and the ways they differ from adjacent forest ecosystems in environmental functions have not been well characterized. Our study investigated family-level floristic and soil differences between adjacent paired patches of intact waterlogged peat forests and kerangas (free-draining heath) forests in Brunei Darussalam. For each patch, we examined total and labile nutrient concentrations in soils, tree stand diversity and structural characteristics, functional traits of live leaves and leaf litter, and nutrient resorption during leaf senescence. We found that total nutrients were more abundant in peat and kerangas humus than in kerangas sand, while available nutrients were highest in kerangas humus, suggesting that anoxic conditions in peat soils impair mineralization of nutrients to available forms but do not lead to losses of nutrient capital. We also found significant compositional differences among those families that occur frequently in both peat and kerangas plots. Despite this, family-level measures of tree diversity and structural characteristics, including tree abundance and stand basal area, did not differ between forest types. Similarly, leaf and litter functional traits and nutrient resorption were invariant across forest types, indicating low plasticity of leaf characteristics associated with plant nutrition. This suggests that belowground carbon accumulation in peatlands is disconnected from aboveground plant community characteristics and is likely driven by belowground processes.

Acteoside is a prominent phenylethanoid glycoside (PhG) with diverse pharmacological activities. However, its chemical synthesis has been challenging due to the reliance on extensive protection/deprotection strategies, leading to lengthy routes and low overall yields. Herein, we present a streamlined and efficient synthetic approach that minimizes synthetic complexity while improving overall efficiency. The strategy, which gave acteoside in 18.6 % overall yield over just 6 steps, employs key regio‐ and chemoselective transformations, including β‐glycosylation, selective caffeoylation, regioselective silylation, α‐rhamnosylation, and a one‐pot global deprotection. By exploiting the inherent differences in hydroxyl reactivity, this method significantly reduces the need for protecting groups, ensuring a more direct synthetic pathway. Importantly, the approach prevents E : Z isomerization of the caffeoyl moiety, preserving the structural integrity of the final product. This methodology can be extended to a broader class of phenylethanoid glycosides, facilitating access to these bioactive natural products for further applications.

Background There is a current, absence of reliable, blood-sparing, diagnostic tools to measure and trend real-time changes in the levels of inflammation and its effects on the immune cells in the infant. Methods We deployed the B iophysica L I mmune P rofiling for I nfants (BLIPI) system in the neonatal intensive care unit to describe immune cell biophysical profiles using 50 microliters of blood per sample from term and preterm infants. Results A total of 19 infants (8 term, 11 preterm) were recruited and 24 blood samples were collected in their first month. Based on the profiles of immune cells’ size and deformation, there was a clear distinction between term and preterm infants, with 48/50 markers significantly different. A preterm infant with late-onset bacterial sepsis had notable size and deformability differences compared to the rest of the preterm cohort. There was a significant correlation between immune cell biophysical profiles and clinical markers such as C-reactive protein, white blood cell counts, and immature-to-total neutrophil (I:T) ratios, with Pearson correlation coefficients for linear regression models of 0.98, 0.97 and 0.94 respectively. Conclusion This study highlights the potential for the biophysical immune cell profiling system to provide an overview of the infant’s current immune activation and response. Impact We present a novel, minimally invasive diagnostic system that leverages the physical properties of immune cells to provide a rapid and direct assessment of the immune status, requiring 20 times less blood volume than standard tests. This study demonstrates the potential of a compact, deployable system that is capable of performing biophysical profiling to assess immune cell activation in term and preterm infants, by revealing distinct differences in cell size and deformation between groups. The system’s sensitive, quantitative measures were correlated with routine clinical biomarkers, highlighting its ability to provide a rapid, minimally invasive, real-time monitoring of neonatal immune status.

We demonstrate the feasibility of machine-learning aided UV absorbance spectroscopy for in-process microbial contamination detection during cell therapy product (CTP) manufacturing. This method leverages a one-class support vector machine to analyse the absorbance spectra of cell cultures and predict if a sample is sterile or contaminated. This label-free technique provides a rapid output (< 30 minutes) with minimal sample preparation and volume (< 1 mL). Spiking of 7 microbial organisms into mesenchymal stromal cells supernatant aliquots from 6 commercial donors showed that contamination events could be detected at low inoculums of 10 CFUs with mean true positive and negative rates of 92.7% and 77.7% respectively. The true negative rate further improved to 92% after excluding samples from a single donor with anomalously high nicotinic acid. In cells spiked with 10 CFUs of E. coli, contamination was detected at the 21-hour timepoint, demonstrating comparable sensitivity to compendial USP < 71 > test (~ 24 hours). We hypothesize that spectral differences between nicotinic acid and nicotinamide in the UV region are the underlying mechanisms for contamination detection. This approach can be deployed as a preliminary test during different CTP manufacturing stages, for real-time, continuous culture monitoring enabling early detection of microbial contamination, assuring safety of CTP.

The remineralization of terrestrial dissolved organic carbon (tDOC) plays an important role in coastal carbon and nutrient cycling, and can affect primary productivity and seawater pH. However, the fate of tDOC in the ocean remains poorly understood. Southeast Asia’s Sunda Shelf Sea receives around 10% of global tDOC input from peatland-draining rivers. Here, we performed photodegradation and long-term (2 months to 1.5 years) biodegradation experiments with samples from peatland-draining rivers and from peat tDOC-rich coastal water. We used the resulting photochemical and microbial decay rates to parameterize a 1-dimensional model simulation. This indicates that 24% and 23% of the initial tDOC entering the Sunda Shelf can be remineralized by pure photo- and pure biodegradation, respectively, after 2 years (which represents an upper limit of seawater residence time on the Sunda Shelf). We also show for the first time that the biodegradation rate of Southeast Asian peat tDOC is enhanced by prior photodegradation. Adding photo-enhanced biodegradation to our model simulation causes remineralization of an additional 16% of the initial tDOC. However, the contribution of photo-enhanced biodegradation was likely underestimated because the photo- and biodegradation steps were conducted successively in our experiments. Overall, our results suggest a notably higher contribution of photodegradation compared with other regions, owing to the combination of slow biodegradation, high solar irradiance, long water residence time on the shelf, and the photo-enhancement of the biodegradation rate. Our results are important for informing tDOC modeling studies, and highlight a need for further research on interactive photo–biodegradation of tDOC. Online view-only full text is available at https://rdcu.be/eaqVS

Queuosine (Q) is a modification of the wobble base in tRNAs that decode NA(C/U) codons. It is ubiquitous in bacteria, including many pathogens. Streptococcus mutans is an early colonizer of dental plaque biofilm and a key player in dental caries. Using a combination of genetic and physiological approaches, the predicted Q synthesis and salvage pathways were validated in this organism. These experiments confirmed that S. mutans can synthesize Q de novo through similar pathways found in Bacillus subtilis and Escherichia coli. However, S. mutans has a distinct salvage pathway compared to these model organisms, as it uses a transporter belonging to the energy coupling factor (ECF) family controlled by a preQ1‐dependent riboswitch. Furthermore, Q levels in this oral pathogen depended heavily on the media composition, suggesting that micronutrients can affect Q‐mediated translation efficiency.

Meat cuts, when cooked and masticated, separate into fibrous structures because of the long-range mechanical anisotropy (LMA) exhibited by muscle fascicles, which is not fully recapitulated in alternative proteins produced using molecular alignment technology like high moisture extrusion. We have developed a scalable perforated micro-imprinting technology to greatly enhance LMA in high moisture meat analogue (HMMA). By imprinting 1 mm thick HMMA sheets with perforated patterns (optimized by AI), we observed up to 5 × more anisotropic separation of fibrous structures in a one-dimensional pulling LMA analysis, to match the fibrousness of the cooked chicken breast, duck breast, pork loin and beef loin. We stacked and bound imprinted sheets with transglutaminase (TG) to produce imprinted whole-cuts. Controlling fiber separation in the imprinted cuts achieved hardness ranging from 6578 g to 18467 g (2 cm × 2 cm × 1 cm, 50% strain), which matched meats from different species. Imprinted cuts improved meat-like fiber separation over HMMA when masticated, measured by Euclidean distances (0.057 and 0.106 respectively) to animal meat cuts on image features. In sensory evaluation, imprinted cuts improved consumer acceptance by 33.3% and meat-like fibrousness by 20%, by significantly enhancing the HMMA appearance, texture, and mouthfeel.

Host-parasite relationships drive the evolution of both parties. In microbe-phage dynamics, CRISPR functions as an adaptive defense mechanism, updating immunity via spacer acquisition. Here, we investigated these interactions within the human gut microbiome, uncovering low frequencies of spacer acquisition at an average rate of one spacer every ∼2.9 point mutations using isolates’ whole genomes and ∼2.7 years using metagenome time series. We identified a highly prevalent CRISPR array in Bifidobacterium longum spreading via horizontal gene transfer (HGT), with six spacers found in various genomic regions in 15 persons from the United States and Europe. These spacers, targeting two prominent Bifidobacterium phages, comprised 76% of spacer occurrence of all spacers targeting these phages in all B. longum populations. This result suggests that HGT of an entire CRISPR-Cas system introduced three times more spacers than local CRISPR-Cas acquisition in B. longum. Overall, our findings identified key ecological and evolutionary factors in prokaryote adaptive immunity.

Quiescent cells require a continuous supply of proteins to maintain protein homeostasis. In fission yeast, entry into quiescence is triggered by nitrogen stress, leading to the inactivation of TORC1 and the activation of TORC2. In this study, we demonstrate that the Greatwall-Endosulfine-PPA/B55 pathway connects the downregulation of TORC1 with the upregulation of TORC2, resulting in the activation of Elongator-dependent tRNA modifications crucial for sustaining the translation programme during entry into quiescence. This mechanism promotes U34 and A37 tRNA modifications at the anticodon stem loop, enhancing translation efficiency and fidelity of mRNAs enriched for AAA versus AAG lysine codons. Notably, several of these mRNAs encode TORC1 inhibitors, TORC2 activators, tRNA modifiers, and proteins necessary for telomeric and subtelomeric functions. Therefore, we propose a mechanism by which cells respond to nitrogen stress at the level of translation, involving a coordinated interplay between tRNA epitranscriptome and biased codon usage.

Background The emergence of induced pluripotent stem cells (iPSCs) offers a promising approach for replacing damaged neurons and glial cells, particularly in spinal cord injuries (SCI). Despite its merits, iPSC differentiation into spinal cord progenitor cells (SCPCs) is variable, necessitating reliable assessment of differentiation and validation of cell quality and safety. Phenotyping is often performed via label-based methods including immunofluorescent staining or flow cytometry analysis. These approaches are often expensive, laborious, time-consuming, destructive, and severely limits their use in large scale cell therapy manufacturing settings. On the other hand, cellular biophysical properties have demonstrated a strong correlation to cell state, quality and functionality and can be measured with ingenious label-free technologies in a rapid and non-destructive manner. Method In this study, we report the use of Magnetic Resonance Relaxometry (MRR), a rapid and label-free method that indicates iron levels based on its readout (T2). Briefly, we differentiated human iPSCs into SCPCs and compared key iPSC and SCPC cellular markers to their intracellular iron content (Fe³⁺) at different stages of the differentiation process. Results With MRR, we found that intracellular iron of iPSCs and SCPCs were distinctively different allowing us to accurately reflect varying levels of residual undifferentiated iPSCs (i.e., OCT4⁺ cells) in any given population of SCPCs. MRR was also able to predict Day 10 SCPC OCT4 levels from Day 1 undifferentiated iPSC T2 values and identified poorly differentiated SCPCs with lower T2, indicative of lower neural progenitor (SOX1) and stem cell (Nestin) marker expression levels. Lastly, MRR was able to provide predictive indications for the extent of differentiation to Day 28 spinal cord motor neurons (ISL-1/SMI-32) based on the T2 values of Day 10 SCPCs. Conclusion MRR measurements of iPSCs and SCPCs has clearly indicated its capabilities to identify and quantify key phenotypes of iPSCs and SCPCs for end-point validation of safety and quality parameters. Thus, our technology provides a rapid label-free method to determine critical quality attributes in iPSC-derived progenies and is ideally suited as a quality control tool in cell therapy manufacturing.

This Letter reports a double heterostructure (DH) AlN/GaN/AlGaN-on-Si HEMT, which has been proposed, for low voltage (LV, ≤5 V) RF operation. The proposed transistor shows excellent DC ( I_dmax =1.9 A/mm, g_mmax =0.66 S/mm) and RF small-signal characteristics ( f_T/f_max =145/195 GHz). Continuous-wave (CW) load-pull measurements at 30 GHz yield P_sat of 0.6 (1.3) W/mm at V_ds of 3.5 (5) V, and peak power-added efficiency (PAE) of 43% (42%). To the best of the authors’ knowledge, the P_sat values are the highest reported for LV GaN-on-Si HEMTs in 5G FR2, despite the use of conventional alloyed contacts and a gate length ( L_g ) of 120 nm. Furthermore, among published LV GaN-on-Si HEMTs, the proposed transistor achieves a desired combination of saturation velocity ( v_sat ) and knee voltage ( V_knee ), which are critical factors for LV power amplification. The results reflect the promising potential of the proposed heterostructure to achieve high transmit power in 5G FR2 handsets.

Nucleic acid amplification tests (NAATs) have enabled fast and sensitive detection of virus infections but are unable to discriminate between live and dead/inert viral fragments or between latent and reactivated virus infections. Here, we show that extracellular viral microRNAs (viral exmiRs) are cell-free candidate biomarkers of live, latent, and reactivated virus infections, achieving fast (under 1 day) and sensitive (30 attomolar [aM]) detection by quantitative real-time reverse transcription PCR (real-time RT-qPCR). We report that spent-media-derived Epstein-Barr virus (EBV) miR-BART10-3p and herpes simplex virus 1 (HSV-1) miR-H5 are biomarkers of live EBV-2 and HSV-1 infection of T cell cultures, respectively. We identified extracellular human herpesvirus 6 (HHV-6) miR-Ro6-4 as a biomarker of endogenous latent HHV-6 in healthy human donor T cell cultures and identified human cytomegalovirus (HCMV) miR-US5-2-5p and miR-US22-5p as plasma biomarkers of endogenous latent HCMV infection. Viral exmiR profiling of spent media from EBV- and HHV-8-reactivated B cell models revealed specific signatures of elevated EBV miR-BHRF1-2-3p and HHV-8 miR-K12-10a-3p, miR-K12-10b, and miR-K12-12-3p, respectively, during virus reactivation. Our study thus suggests the utility of viral exmiR biomarkers in enabling NAAT-based detection of live, endogenous latent, and reactivated virus infections of cells.