Biology subjects

Neff, R.

Publications and source records attributed to Neff, R..

5 recordsLinked to original sources

bioRxiv2026

Cyanophage CP12 Rewires Host Carbon Regulation through Interface Remodeling and Redox Buffering

Picocyanobacteria drive ocean carbon fixation, and cyanophages reshape host metabolism during infection. In cyanobacteria, the intrinsically disorder Calvin cycle protein 12 (CP12) assembles glyceraldehyde-3-phosphate dehydrogenase (GAP2) and phosphoribulokinase (PRK) into the inhibitory dark complex, yet how phage CP12 homologs modulate redox-sensitive partners remains unclear. Here, we examined viral CP12 across sequence, structure, and post-translational modification (PTM) complexities to resolve the mechanistic remodeling of dark-complex assembly and regulation. Protein-family analysis of CP12 homologs across diverse lineages showed that viral CP12 preserves interface-dominated positions while shifting partner-facing chemistry toward charged and geometry-modulating features. Matched molecular dynamics simulations (MD) of Prochlorococcus MED4 and cyanophage P-HM2 showed that phage CP12 preserves assembly while strengthening PRK-facing contacts and reducing GAP2-facing interface burial. MED4 redox proteomics identified coordinated cysteine oxidation across CP12 and GAP2 under light disturbance, guiding MD to explore thiol-PTM states. Conformational divergence increased with PTM load and localized mainly to GAP2 modifications. Thiol PTM at GAP2 imposed the largest CP12 binding-energy cost, which P-HM2 CP12 buffered, yielding smaller comparative binding-energy penalties than host CP12. These findings link sequence-driven chemistry to interface dynamics and redox PTM responsiveness to light, defining phage CP12 as a regulatory mimetic that may retune host carbon regulation during infection.

microbiology↗

bioRxiv2026

Real-time electrochemical protein monitoring using molecular pendulum sensors

Continuous monitoring of proteins in complex biological fluids is essential for advancing personalized medicine, yet existing biosensors are often limited by instability, single-use designs, and insufficient sensitivity. Here, we describe a detailed protocol for the fabrication and operation of molecular pendulum (MP) electrochemical sensors integrated with an active-reset mechanism to enable real-time, reversible, and ultrasensitive protein detection. The protocol is described in two parts. First, we describe the microfabrication of gold microelectrodes and their nanostructured modification, followed by assembly of DNA-based pendulum probes with redox reporters and affinity receptors that translate binding into kinetic electron-transfer shifts. Second, we detail the sensing and active-reset approach, which detects the target analyte and applies tunable oscillatory potentials to accelerate its dissociation, regenerate sensor surfaces, and extend operational lifetime. The protocol includes detailed guidance on device fabrication, surface functionalization, sensing and reset cycles, and data analysis. When implemented, MP sensors with active-reset achieve pg/ml sensitivity, rapid equilibration, and robust performance across biofluids and in situ models, enabling continuous protein monitoring over extended periods. This combined technology represents a biosensing platform with significant potential, opening new avenues for wearable and implantable molecular monitoring, early disease detection, and personalized therapeutic guidance.

bioengineering↗

bioRxiv2025

Myelin Mapping in the Human Brain Using an Empirical Extension of the Ridge Regression Theorem

PurposeMyelin water fraction (MWF) mapping in the central nervous system is a topic of intense research activity. One framework for this requires parameter estimation from a decaying biexponential signal. However, this is often an ill-posed nonlinear problem resulting in unreliable parameter estimates. For linear least-squares (LLS) problems, the ridge regression theorem (RRT) shows that a Tikhonov regularization parameter exists that will reduce mean square error (MSE) in parameter estimates. We present and apply a nonlinear version of the RRT,{lambda} -NL-RR, to MWF mapping. MethodsFor simulated and experimental data, we estimated parameter values with conventional nonlinear least-squares (NLLS) and compared these with values obtained from{lambda} -NL-RR, with the regularization parameter value defined by generalized cross validation. We applied regularization only to signals identified as biexponential according to the Bayesian information criterion. ResultsUnder conditions of modest SNR and closely spaced exponential time constants in which conventional biexponential analysis methods yield particularly inaccurate results,{lambda} -NL-RR decreases MSE by ~10-15%. ConclusionRegularization of the NLLS parameter estimation problem for the biexponential model decreased MSE for simulated and in vivo MRI brain data. In addition, this work provides a general framework for regularization of a broad class of NLLS problems.

biophysics↗

bioRxiv2024

Advanced Tissue Technologies of Blood-Brain Barrier Organoids as High Throughput Toxicity Readouts in Drug Development

Recent advancements in engineering Complex in vitro models (CIVMs) such as Blood-brain barrier (BBB) organoids offer promising platforms for preclinical drug testing. However, their application in drug development, and especially for the regulatory purposes of toxicity assessment, requires robust and reproducible techniques. Here, we developed an adapted set of orthogonal image-based tissue methods including hematoxylin and eosin staining (HE), immunohistochemistry (IHC), multiplex immunofluorescence (mIF), and Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI-MSI) to validate CIVMs for drug toxicity assessments. We developed an artificial intelligence (AI) algorithm to increase the throughput and the reliability of histomorphologic evaluations of apoptosis for in vitro toxicity studies. Our data highlight the potential to integrate advanced morphology-based readouts such as histological techniques and digital pathology algorithms for use on CIVMs, as part of a standard preclinical drug development assessment. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/611987v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@190530eorg.highwire.dtl.DTLVardef@3cce5forg.highwire.dtl.DTLVardef@10ec3borg.highwire.dtl.DTLVardef@1051585_HPS_FORMAT_FIGEXP M_FIG C_FIG The graphical abstract was partially created with biorender.com. HighlightsO_LIAdvanced Complex in vitro models (CIVMs) like Blood-brain barrier (BBB) organoids show promise for preclinical drug testing. However, robust and reproducible techniques are crucial for the acceptance of CIVMs in drug development processes, especially for toxicity assessments which are highly regulated by health authorities. C_LIO_LIWe developed orthogonal image-based readouts on histological sections to enable the use of BBB organoids for future compound toxicity assessment. C_LIO_LIA newly established artificial intelligence (AI) algorithm provides automated and label-free detection of apoptotic cells in drug screening using BBB organoids and provides an alternative killing assay on single cell resolution (40x) to current standards. C_LI

pathology↗

bioRxiv2019

Molecular Networks and Key Regulators of the Dysregulated Neuronal System in Alzheimer’s Disease

To study the molecular mechanisms driving the pathogenesis and identify novel therapeutic targets of late onset Alzheimers Disease (LOAD), we performed an integrative network analysis of whole-genome DNA and RNA sequencing profiling of four cortical areas, including the parahippocampal gyrus, across 364 donors spanning the full spectrum of LOAD-related cognitive and neuropathological disease severities. Our analyses revealed thousands of molecular changes and uncovered for the first-time multiple neuron specific gene subnetworks most dysregulated in LOAD. ATP6V1A, a critical subunit of vacuolar-type H+-ATPase (v-ATPase), was predicted to be a key regulator of one neuronal subnetwork and its role in disease-related processes was evaluated through CRISPR-based manipulation of human induced pluripotent stem cell derived neurons and RNAi-based knockdown in transgenic Drosophila models. This study advances our understanding of LOAD pathogenesis by providing the global landscape and detailed circuits of complex molecular interactions and regulations in several key brain regions affected by LOAD and the resulting network models provide a blueprint for developing next generation therapeutics against LOAD.

neuroscience↗