Biology subjects

Ritchie, A.

Publications and source records attributed to Ritchie, A..

5 recordsLinked to original sources

bioRxiv2026

Regulation of RNA maturation by the family of human G-patch proteins

The family of human G-patch proteins comprises more than 20 members, each characterized by a glycine-rich G-patch implicated in mediating interactions with RNA helicases. Here, we systematically identify the cognate RNA helicase of each G-patch protein, highlighting the association of DHX15 with a network of 20 G-patch cofactors. DHX35 and GPATCH1 represent a unique G-patch protein-RNA helicase pair, and we uncover a regulatory circuit between these partners. Comprehensive in vitro analyses of ATPase activity and RNA binding identify distinguishing features of DHX15- and non-DHX15-associated G-patch proteins, and demonstrate the roles of most G-patch proteins as bona fide stimulatory cofactors of DHX15. RNA interactome analyses of each G-patch protein and complementary transcriptome-wide alternative splicing analyses in cells lacking a G-patch protein reveal distinct modes of regulation of mRNA maturation by different G-patch proteins. For example, ZGPAT affects splicing indirectly through its requirement for efficient 2'-O-methylation of snRNAs, GPATCH8 exemplifies DHX15-associated alternative splicing modulation, whereas SUGP2 suppresses splicing in an RNA helicase-independent manner via direct binding to pre-mRNA introns.

molecular biology↗

bioRxiv2026

IMPAIRED BRIDGING OF TEMPORAL DISCONTINUITIES IN OLDER ADULT HIV-1 TG RATS

The advent and widespread uptake of combination antiretroviral therapy dramatically changed the epidemiological features of human immunodeficiency virus type 1 (HIV-1), whereby older individuals (>50 years of age) account for approximately 50% of HIV-1 seropositive individuals in the United States. Nevertheless, to date, there is no extant in vivo biological system to model the unique age-related neurocognitive impairments observed in HIV-1 seropositive individuals. Herein, the utility of the HIV-1 transgenic (Tg) rat as a biological system to model age-related neurocognitive impairments and neuroanatomical alterations was evaluated. Older adult HIV-1 Tg rodents (i.e., >12 months of age upon testing initiation), relative to their control counterparts, exhibited profound neurocognitive alterations characterized by impairments in stimulus-reinforcement learning, sustained attention, and selective attention; neurocognitive deficits which support a fundamental distortion of temporal processing. Neuronal dysfunction in older adult HIV-1 Tg animals was characterized by structural alterations in pyramidal neurons, and their associated dendritic spines, in the medial prefrontal cortex and abnormal accumulation of amyloid beta (A{beta}). Interestingly, the abnormal accumulation of A{beta} mechanistically underlies, at least in part, the profound dendritic spine dysmorphology in male, but not female, HIV-1 Tg rats. More critically, however, neuronal dysfunction mechanistically underlies neurocognitive impairments in both male and female HIV-1 Tg rodents, whereby neuronal dysfunction accounts for 65.4% and 60.8% of the variance in neurocognitive function, respectively. Establishing the utility of the HIV-1 Tg rat for age-related neurocognitive impairments is fundamental to disentangling the role of HIV-1 viral proteins and comorbidities in neurocognitive function.

neuroscience↗

bioRxiv2026

Optimization of isolation, expansion, and differentiation of canine intestinal organoids

Intestinal organoids are three-dimensional in vitro structures derived from stem cells and serve as a valuable model for studying intestinal biology and pathophysiology. This study optimized the isolation, expansion, and differentiation of canine intestinal organoids from duodenum and colon. Organoids were generated from canine intestinal crypts and cultured in Matrigel with a growth factor cocktail. The impact of prostaglandin E2 (PGE2) concentration on organoid growth was evaluated, and a two-phase differentiation protocol--comprising patterning and differentiation media--was implemented, including interleukin (IL)-22 in the duodenal differentiation phase. Organoids cultured with 100 nM PGE2 exhibited increased crypt budding and organoid-forming efficiency, indicative of enhanced stem cell proliferation. Differentiated organoids expressed key intestinal markers (VIL1, SI, CHGA, MUC2), and forskolin-induced swelling demonstrated functional Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) activity. Although the sample size (n=2) limits generalizability, this optimized protocol provides a relevant in vitro model for studying canine intestinal function. The model can be used in future research for disease modelling and translational applications, supporting downstream studies in gastrointestinal disease, drug permeability, and comparative One Health research.

cell biology↗

bioRxiv2024

Tumour-derived LAMA5 is critical for tumour initiation and controls progression and phenotype in luminal breast cancer

Basement membrane (BM) supports and regulates the structural integrity, function and differentiation of epithelial tissues and protects against breast cancer invasion to stroma. Here we show that the BM component LAMA5 is critical for initiation of luminal mammary tumours, and controls tumour progression and phenotype development. LAMA5 is overexpressed in human breast carcinomas and LAMA5 downregulation attenuates growth of human breast cancer cells. Prepubertal luminal deletion of Lama5 in MMTV-PyMT mice results in marked reduction in emergence of early hyperplasias, along with a shift towards luminal progenitor-like phenotype. However, single allele deletion, but not biallelic deletion of Lama5 inhibits growth and progression towards advanced mammary carcinomas. Lama5 deficient luminal epithelial cells collectively display widespread alterations in Fibroblast growth factor (Fgf) signalling genes, including increased expression of Fgf receptor 2 (Fgfr2). Inhibition of Fgf receptors decreases growth and induces apoptosis also in biallelic-Lama5-deleted organoids without affecting wildtype organoids. Our results demonstrate a critical role for the BM component LAMA5 in mammary tumour initiation and reveal mechanisms of ECM-epithelial interplay in breast tumour progression and phenotype maintenance.

cancer biology↗

bioRxiv2024

Mass Generation and Long-term Expansion of Hepatobiliary Organoids from Adult Primary Human Hepatocytes

Adult primary human hepatocytes (PHHs) are the gold standard in ex vivo toxicological studies and possess the clinical potential to treat patients with liver disease as advanced therapy medicinal products (ATMPs). However, the utility of this valuable cell type has been limited by short-term functionality and limited expansion potential in vitro. While notable advances have been made in the long-term maintenance of primary hepatocytes, there has been limited success in driving the efficient generation and expansion of adult PHH-derived organoids which recapitulate both liver tissue architecture and function, hampering in vitro studies and regenerative medicine applications. Here we describe the mass generation and long-term expansion of hepatobiliary organoids with functionally interconnected hepatic and biliary-like structures from adult primary human hepatocytes. Hepatobiliary organoids retain the expression of lineage and functional markers, closely resembling PHH, while also acquiring the expression of regeneration, fetal and biliary markers. Organoids perform key hepatocyte functions while proliferating and can be matured to enhance their functionality. As a proof-of-principle, we demonstrate that hepatobiliary organoids can recapitulate hallmarks of cholestasis and steatosis in vitro. Moreover, we show that hepatocytes can be transfected, transduced and gene edited in 3D prior to organoid generation, facilitating a wide range of applications. Our novel hepatobiliary organoid system bridges the gap between short-term functionality of primary human hepatocytes and the need for scalable, long-term organoid models of the adult liver, offering immense potential for drug testing, disease modeling, and advanced therapeutic applications.

cell biology↗