A tale of two waves: Delineating diverse genomic and transmission landscapes driving the COVID-19 pandemic in Pune, India.

BACKGROUND: Modern response to pandemics, critical for effective public health measures, is shaped by the availability and integration of diverse epidemiological outbreak data. Tracking variants of concern (VOC) is integral to understanding the evolution of SARS-CoV-2 in space and time, both at the local level and global context. This potentially generates actionable information when integrated with epidemiological outbreak data. METHODS: A city-wide network of researchers, clinicians, and pathology diagnostic laboratories was formed for genome surveillance of COVID-19 in Pune, India. The genomic landscapes of 10,496 sequenced samples of SARS-CoV-2 driving peaks of infection in Pune between December-2020 to March-2022, were determined. As a modern response to the pandemic, a "band of five" outbreak data analytics approach was used. This integrated the genomic data (Band 1) of the virus through molecular phylogenetics with key outbreak data including sample collection dates and case numbers (Band 2), demographics like age and gender (Band 3-4), and geospatial mapping (Band 5). RESULTS: The transmission dynamics of VOCs in 10,496 sequenced samples identified B.1.617.2 (Delta) and BA(x) (Omicron formerly known as B.1.1.529) variants as drivers of the second and third peaks of infection in Pune. Spike Protein mutational profiling during pre and post-Omicron VOCs indicated differential rank ordering of high-frequency mutations in specific domains that increased the charge and binding properties of the protein. Time-resolved phylogenetic analysis of Omicron sub-lineages identified a highly divergent BA.1 from Pune in addition to recombinant X lineages, XZ, XQ, and XM. CONCLUSIONS: The band of five outbreak data analytics approach, which integrates five different types of data, highlights the importance of a strong surveillance system with high-quality meta-data for understanding the spatiotemporal evolution of the SARS-CoV-2 genome in Pune. These findings have important implications for pandemic preparedness and could be critical tools for understanding and responding to future outbreaks.

Antagonistic Activities of Fmn2 and ADF Regulate Axonal F-Actin Patch Dynamics and the Initiation of Collateral Branching.

Interstitial collateral branching of axons is a critical component in the development of functional neural circuits. Axon collateral branches are established through a series of cellular processes initiated by the development of a specialized, focal F-actin network in axons. The formation, maintenance and remodeling of this F-actin patch is critical for the initiation of axonal protrusions that are subsequently consolidated to form a collateral branch. However, the mechanisms regulating F-actin patch dynamics are poorly understood. Fmn2 is a formin family member implicated in multiple neurodevelopmental disorders. We find that Fmn2 regulates the initiation of axon collateral protrusions in chick spinal neurons and in zebrafish motor neurons. Fmn2 localizes to the protrusion-initiating axonal F-actin patches and regulates the lifetime and size of these F-actin networks. The F-actin nucleation activity of Fmn2 is necessary for F-actin patch stability but not for initiating patch formation. We show that Fmn2 insulates the F-actin patches from disassembly by the actin-depolymerizing factor, ADF, and promotes long-lived, larger patches that are competent to initiate axonal protrusions. The regulation of axonal branching can contribute to the neurodevelopmental pathologies associated with Fmn2 and the dynamic antagonism between Fmn2 and ADF may represent a general mechanism of formin-dependent protection of Arp2/3-initiated F-actin networks from disassembly.SIGNIFICANCE STATEMENT Axonal branching is a key process in the development of functional circuits and neural plasticity. Axon collateral branching is initiated by the elaboration of F-actin filaments from discrete axonal F-actin networks. We show that the neurodevelopmental disorder-associated formin, Fmn2, is a critical regulator of axon collateral branching. Fmn2 localizes to the collateral branch-inducing F-actin patches in axons and regulates the stability of these actin networks. The F-actin nucleation activity of Fmn2 protects the patches from ADF-mediated disassembly. Opposing activities of Fmn2 and ADF exert a dynamic regulatory control on axon collateral branch initiation and may underly the neurodevelopmental defects associated with Fmn2.

Synthesis of Phosphorodiamidate Morpholino Oligonucleotides Using Trityl and Fmoc Chemistry in an Automated Oligo Synthesizer.

Phosphorodiamidate morpholino oligonucleotides (PMOs) constitute 3 out of the 11 FDA-approved oligonucleotide-based drugs in the last 6 years. PMOs can effectively silence disease-causing genes and modify splicing. However, PMO synthesis has remained challenging for a variety of reasons: inefficient deprotection and coupling methods and instability of monomers. Here, we report the development of a suitable combination of resin supports, deblocking and coupling reagents for synthesizing PMOs using either trityl or Fmoc-protected chlorophosphoramidate monomers. The synthesized PMOs using both the methods on a solid support have been validated for gene silencing in a zebrafish model. The protocol was successfully transferred into an automated DNA synthesizer to make several sequences of PMOs, demonstrating for the first time the adaptation of regular PMOs in a commercial DNA synthesizer. Moreover, PMOs with longer than 20-mer sequences, including FDA-approved Eteplirsen (30-mer), were achieved in >20% overall yield that is superior to previous reports. Hybridization study shows that PMOs exhibit a higher binding affinity toward complementary DNA relative to the DNA/DNA duplex (>6 °C). Additionally, the introduction of Fmoc chemistry into PMOs opens up the possibility for PMO synthesis in commercial peptide synthesizers for future development.

The Formin Fmn2b Is Required for the Development of an Excitatory Interneuron Module in the Zebrafish Acoustic Startle Circuit.

The formin family member Fmn2 is a neuronally enriched cytoskeletal remodeling protein conserved across vertebrates. Recent studies have implicated Fmn2 in neurodevelopmental disorders, including sensory processing dysfunction and intellectual disability in humans. Cellular characterization of Fmn2 in primary neuronal cultures has identified its function in the regulation of cell-substrate adhesion and consequently growth cone translocation. However, the role of Fmn2 in the development of neural circuits in vivo, and its impact on associated behaviors have not been tested. Using automated analysis of behavior and systematic investigation of the associated circuitry, we uncover the role of Fmn2b in zebrafish neural circuit development. As reported in other vertebrates, the zebrafish ortholog of Fmn2 is also enriched in the developing zebrafish nervous system. We find that Fmn2b is required for the development of an excitatory interneuron pathway, the spiral fiber neuron, which is an essential circuit component in the regulation of the Mauthner cell (M-cell)-mediated acoustic startle response. Consistent with the loss of the spiral fiber neurons tracts, high-speed video recording revealed a reduction in the short latency escape events while responsiveness to the stimuli was unaffected. Taken together, this study provides evidence for a circuit-specific requirement of Fmn2b in eliciting an essential behavior in zebrafish. Our findings underscore the importance of Fmn2 in neural development across vertebrate lineages and highlight zebrafish models in understanding neurodevelopmental disorders.

Coupling of dynamic microtubules to F-actin by Fmn2 regulates chemotaxis of neuronal growth cones.

Dynamic co-regulation of the actin and microtubule subsystems enables the highly precise and adaptive remodelling of the cytoskeleton necessary for critical cellular processes, such as axonal pathfinding. The modes and mediators of this interpolymer crosstalk, however, are inadequately understood. We identify Fmn2, a non-diaphanous-related formin associated with cognitive disabilities, as a novel regulator of cooperative actin-microtubule remodelling in growth cones of both chick and zebrafish neurons. We show that Fmn2 stabilizes microtubules in the growth cones of cultured spinal neurons and in vivo. Super-resolution imaging revealed that Fmn2 facilitates guidance of exploratory microtubules along actin bundles into the chemosensory filopodia. Using live imaging, biochemistry and single-molecule assays, we show that a C-terminal domain in Fmn2 is necessary for the dynamic association between microtubules and actin filaments. In the absence of the cross-bridging function of Fmn2, filopodial capture of microtubules is compromised, resulting in destabilized filopodial protrusions and deficits in growth cone chemotaxis. Our results uncover a critical function for Fmn2 in actin-microtubule crosstalk in neurons and demonstrate that the modulation of microtubule dynamics via associations with F-actin is central to directional motility.