PTEN inhibition promotes robust growth of bulbospinal respiratory axons and partial recovery of diaphragm function in a chronic model of cervical contusion spinal cord injury.

High spinal cord injury (SCI) leads to persistent and debilitating compromise in respiratory function. Cervical SCI not only causes the death of phrenic motor neurons (PhMNs) that innervate the diaphragm, but also damages descending respiratory pathways originating in the rostral ventral respiratory group (rVRG) located in the brainstem, resulting in denervation and consequent silencing of spared PhMNs located caudal to injury. It is imperative to determine whether interventions targeting rVRG axon growth and respiratory neural circuit reconnection are efficacious in chronic cervical contusion SCI, given that the vast majority of individuals are chronically-injured and most cases of SCI involve contusion-type damage to the cervical region. We therefore employed a rat model of chronic cervical hemicontusion to test therapeutic manipulations aimed at reconstructing damaged rVRG-PhMN-diaphragm circuitry to achieve recovery of respiratory function. At a chronic time point post-injury, we systemically administered: an antagonist peptide directed against phosphatase and tensin homolog (PTEN), a central inhibitor of neuron-intrinsic axon growth potential; an antagonist peptide directed against receptor-type protein tyrosine phosphatase sigma (PTPσ), another important negative regulator of axon growth capacity; or a combination of these two peptides. PTEN antagonist peptide (PAP4) promoted partial recovery of diaphragm motor activity out to nine months post-injury (though this effect depended on the anesthetic regimen used during recording), while PTPσ peptide did not impact diaphragm function after cervical SCI. Furthermore, PAP4 promoted robust growth of descending bulbospinal rVRG axons caudal to the injury within the denervated portion of the PhMN pool, while PTPσ peptide did not affect rVRG axon growth at this location that is critical to control of diaphragmatic respiratory function. In conclusion, we find that, when PTEN inhibition is targeted at a chronic time point following cervical contusion, our non-invasive PAP4 strategy can successfully promote significant regrowth of damaged respiratory neural circuitry and also partial recovery of diaphragm motor function.

PTEN inhibition promotes robust growth of bulbospinal respiratory axons and partial recovery of diaphragm function in a chronic model of cervical contusion spinal cord injury.

High spinal cord injury (SCI) leads to persistent and debilitating compromise in respiratory function. Cervical SCI not only causes the death of phrenic motor neurons (PhMNs) that innervate the diaphragm, but also damages descending respiratory pathways originating in the rostral ventral respiratory group (rVRG) located in the brainstem, resulting in denervation and consequent silencing of spared PhMNs located caudal to injury. It is imperative to determine whether interventions targeting rVRG axon growth and respiratory neural circuit reconnection are efficacious in chronic cervical contusion SCI, given that the vast majority of individuals are chronically-injured and most cases of SCI involve contusion-type damage to the cervical region. We therefore employed a clinically-relevant rat model of chronic cervical hemicontusion to test therapeutic manipulations aimed at reconstructing damaged rVRG-PhMN-diaphragm circuitry to achieve recovery of respiratory function. At a chronic time point post-injury, we systemically administered: an antagonist peptide directed against phosphatase and tensin homolog (PTEN), a central inhibitor of neuron-intrinsic axon growth potential; an antagonist peptide directed against receptor-type protein tyrosine phosphatase sigma (PTPσ), another important negative regulator of axon growth capacity; or a combination of these two peptides. PTEN antagonist peptide (PAP4) promoted partial recovery of diaphragm motor activity out to nine months post-injury, while PTPσ peptide did not impact diaphragm function after cervical SCI. Furthermore, PAP4 promoted robust growth of descending bulbospinal rVRG axons caudal to the injury within the denervated portion of the PhMN pool, while PTPσ peptide did not affect rVRG axon growth at this location that is critical to control of diaphragmatic respiratory function. In conclusion, we find that, when PTEN inhibition is targeted at a chronic time point following cervical contusion that is most relevant to the SCI clinical population, our non-invasive PAP4 strategy can successfully promote significant regrowth of damaged respiratory neural circuitry and also partial recovery of diaphragm motor function. HIGHLIGHTS: PTEN antagonist peptide promotes partial diaphragm function recovery in chronic cervical contusion SCI.PTPσ inhibitory peptide does not impact diaphragm function recovery in chronic cervical contusion SCI.PTEN antagonist peptide promotes growth of bulbospinal rVRG axons in chronic cervical contusion SCI.PTPσ peptide does not affect rVRG axon growth in chronic cervical contusion SCI.

Genomic Characterization of Three Novel Bartonella Strains in a Rodent and Two Bat Species from Mexico.

Rodents and bats are the most diverse mammal group that host Bartonella species. In the Americas, they were described as harboring Bartonella species; however, they were mostly characterized to the genotypic level. We describe here Bartonella isolates obtained from blood samples of one rodent (Peromyscus yucatanicus from San José Pibtuch, Yucatan) and two bat species (Desmodus rotundus from Progreso, and Pteronotus parnellii from Chamela-Cuitzmala) from Mexico. We sequenced and described the genomic features of three Bartonella strains and performed phylogenomic and pangenome analyses to decipher their phylogenetic relationships. The mouse-associated genome was closely related to Bartonella vinsonii. The two bat-associated genomes clustered into a single distinct clade in between lineages 3 and 4, suggesting to be an ancestor of the rodent-associated Bartonella clade (lineage 4). These three genomes showed <95% OrthoANI values compared to any other Bartonella genome, and therefore should be considered as novel species. In addition, our analyses suggest that the B. vinsonii complex should be revised, and all B. vinsonii subspecies need to be renamed and considered as full species. The phylogenomic clustering of the bat-associated Bartonella strains and their virulence factor profile (lack of the Vbh/TraG conjugation system remains of the T4SS) suggest that it should be considered as a new lineage clade (L5) within the Bartonella genus.

Response of Astrocyte Subpopulations Following Spinal Cord Injury.

There is growing appreciation for astrocyte heterogeneity both across and within central nervous system (CNS) regions, as well as between intact and diseased states. Recent work identified multiple astrocyte subpopulations in mature brain. Interestingly, one subpopulation (Population C) was shown to possess significantly enhanced synaptogenic properties in vitro, as compared with other astrocyte subpopulations of adult cortex and spinal cord. Following spinal cord injury (SCI), damaged neurons lose synaptic connections with neuronal partners, resulting in persistent functional loss. We determined whether SCI induces an enhanced synaptomodulatory astrocyte phenotype by shifting toward a greater proportion of Population C cells and/or increasing expression of relevant synapse formation-associated genes within one or more astrocyte subpopulations. Using flow cytometry and RNAscope in situ hybridization, we found that astrocyte subpopulation distribution in the spinal cord did not change to a selectively synaptogenic phenotype following mouse cervical hemisection-type SCI. We also found that spinal cord astrocytes expressed synapse formation-associated genes to a similar degree across subpopulations, as well as in an unchanged manner between uninjured and SCI conditions. Finally, we confirmed these astrocyte subpopulations are also present in the human spinal cord in a similar distribution as mouse, suggesting possible conservation of spinal cord astrocyte heterogeneity across species.

Directed Evolution of a Selective and Sensitive Serotonin Sensor via Machine Learning.

Serotonin plays a central role in cognition and is the target of most pharmaceuticals for psychiatric disorders. Existing drugs have limited efficacy; creation of improved versions will require better understanding of serotonergic circuitry, which has been hampered by our inability to monitor serotonin release and transport with high spatial and temporal resolution. We developed and applied a binding-pocket redesign strategy, guided by machine learning, to create a high-performance, soluble, fluorescent serotonin sensor (iSeroSnFR), enabling optical detection of millisecond-scale serotonin transients. We demonstrate that iSeroSnFR can be used to detect serotonin release in freely behaving mice during fear conditioning, social interaction, and sleep/wake transitions. We also developed a robust assay of serotonin transporter function and modulation by drugs. We expect that both machine-learning-guided binding-pocket redesign and iSeroSnFR will have broad utility for the development of other sensors and in vitro and in vivo serotonin detection, respectively.

Facial grimace testing as an assay of neuropathic pain-related behavior in a mouse model of cervical spinal cord injury.

A major portion of individuals affected by traumatic spinal cord injury (SCI) experience one or more types of chronic neuropathic pain (NP), which is often intractable to currently available treatments. The availability of reliable behavioral assays in pre-clinical models of SCI-induced NP is therefore critical to assess the efficacy of new potential therapies. Commonly used assays to evaluate NP-related behavior in rodents, such as Hargreaves thermal and von Frey mechanical testing, rely on the withdrawal response to an evoked stimulus. However, other assays that test spontaneous/non-evoked NP-related behavior or supraspinal aspects of NP would be highly useful for a more comprehensive assessment of NP following SCI. The Mouse Grimace Scale (MGS) is a tool to assess spontaneous, supraspinal pain-like behaviors in mice; however, the assay has not been characterized in a mouse model of SCI-induced chronic NP, despite the critical importance of mouse genetics as an experimental tool. We found that beginning 2 weeks after cervical contusion, SCI mice exhibited increased facial grimace features compared to laminectomy-only control mice, and this grimace phenotype persisted to the chronic time point of 5 weeks post-injury. We also found a significant relationship between facial grimace score and the evoked forepaw withdrawal response in both the Hargreaves and von Frey tests at 5 weeks post-injury when both laminectomy-only and SCI mice were included in the analysis. However, within only the SCI group, there was no correlation between grimace score and Hargreaves or von Frey responses. These results indicate both that facial grimace analysis can be used as an assay of spontaneous NP-related behavior in the mouse model of SCI and that the information provided by the MGS may be different than that provided by evoked tests of sensory function.

Detection of Bartonella infection in pet dogs from Manila, the Philippines.

Dogs can be infected by a wide range of Bartonella spp., but studies regarding the prevalence of Bartonella infection in dogs in the Philippines have not been conducted. This study aimed at determining the prevalence of Bartonella infection in pets dogs from two veterinary clinics in Metro Manila, The Philippines, using both serology and polymerase chain reaction (PCR). Blood samples from 116 dogs from two different groups, one of 60 mainly "healthy dogs" and the other one of 56 dogs enrolled in a tick-borne disease suspect group, were tested for presence of B. henselae antibodies and to detect Bartonella DNA using primers specific for the citrate synthase gene. Seroprevalence for B. henselae was very low (2.6%), as the only three (5%) seropositive dogs (titer 1:64) where among the healthy pet dog group. Following subsequent sequencing, 13 samples, all from the tick-borne disease group, were determined positive for B. henselae (11.2%). This is the first study to report dog infection with B. henselae in the Philippines.

Bartonella Infection in Stray Dogs from Central and Southern Chile (Linares and Puerto Montt).

Bartonellae are emerging zoonotic vector-borne pathogens causing a broad spectrum of clinical symptoms in humans and animals, including life-threatening endocarditis. Dogs are infected with a wide range of Bartonella species and infection has been reported in free-roaming dogs from various South American countries. We report a high Bartonella seroprevalence in 82 Chilean stray dogs. More than half of the dogs from Linares (72.7%, n = 66) and Puerto Montt (56.2%, n = 16) were seropositive for Bartonella henselae, Bartonella vinsonii ssp. berkhoffii, or Bartonella clarridgeiae with antibody titers ranging from 1:64 to 1:512. Three dogs (3.6%) were PCR positive for Bartonella sp. Partial sequencing of the gltA gene indicated that two dogs were infected with B. henselae, and one with a strain close to Bartonella vinsonii ssp. vinsonii. Exposure to Bartonella species was common in stray Chilean dogs, as for other South American countries, likely associated with heavy ectoparasite infestation.

Detection of Bartonella species, including Candidatus Bartonella ovis sp. nov, in ruminants from Mexico and lack of evidence of Bartonella DNA in saliva of common vampire bats (Desmodus rotundus) predating on them.

Bartonella spp. have been identified in many bat species worldwide, including the zoonotic species, Candidatus Bartonella mayotimonensis. The common vampire bat (Desmodus rotundus) preys preferentially on livestock in Latin America and is frequently infected with Bartonella spp. To determine the potential role of D. rotundus in transmitting Bartonella to livestock, common vampire bats and bat-bitten domestic ruminants from Mexico were tested for Bartonella infection by blood culture or conventional PCR. Furthermore, to explore the possibility of bite transmission during blood feeding, saliva swabs from 35 D. rotundus known to be either Bartonella bacteremic (N = 17) or blood culture negative (N = 18) were tested by PCR to detect the presence of Bartonella DNA. Twenty (17.1%) of 117 sheep and 16 (34.8%) of 46 cattle were Bartonella bacteremic by PCR testing. However, none of them were infected with Bartonella strains previously isolated from vampire bats and none of the 35 D. rotundus saliva swabs tested were PCR positive for Bartonella. All but two animals among those which were Bartonella culture and/or PCR positive, were infected with either B. bovis (cattle) or B. melophagi (sheep). Two sheep were infected by a possible new species, Candidatus Bartonella ovis, being phylogenetically closer to B. bovis than B. melophagi. This study does not support the role of D. rotundus as a reservoir of Bartonella species infecting livestock, which could be transmitted via bite and blood feeding and therefore suggest limited risk of zoonotic transmission of Bartonella from common vampire bats to humans.

Molecular Detection of Bartonella Species in Blood-Feeding Bat Flies from Mexico.

Bartonellae are emerging blood-borne bacteria that have been recovered from a wide range of mammalian species and arthropod vectors around the world. Bats are now recognized as a potential wildlife reservoir for a diverse number of Bartonella species, including the zoonotic Candidatus B. mayotimonensis. These bat-borne Bartonella species have also been detected in the obligate ectoparasites of bats, such as blood-feeding flies, which could transmit these bacteria within bat populations. To better understand this potential for transmission, we investigated the relatedness between Bartonella detected or isolated from bat hosts sampled in Mexico and their ectoparasites. Bartonella spp. were identified in bat flies collected on two bat species, with the highest prevalence in Trichobius parasiticus and Strebla wiedemanni collected from common vampire bats (Desmodus rotundus). When comparing Bartonella sequences from a fragment of the citrate synthase gene (gltA), vector-associated strains were diverse and generally close to, but distinct from, those recovered from their bacteremic bat hosts in Mexico. Complete Bartonella sequence concordance was observed in only one bat-vector pair. The diversity of Bartonella strains in bat flies reflects the frequent host switch by bat flies, as they usually do not live permanently on their bat host. It may also suggest a possible endosymbiotic relationship with these vectors for some of the Bartonella species carried by bat flies, whereas others could have a mammalian host.

Bartonella henselae in small Indian mongooses (Herpestes auropunctatus) from Grenada, West Indies.

Many mammals are established hosts for the vector borne bacterial genus, Bartonella. Small Indian mongooses (Herpestes auropunctatus) have only been reported as a possible host for Bartonella henselae in southern Japan. Confirming Bartonella presence in mongooses from other regions in the world may support their role as potential reservoirs of this human pathogen. Specifically, documenting Bartonella in Caribbean mongooses would identify a potential source of zoonotic risk with mongoose-human contact in the New World. Using serological and molecular techniques, we investigated B. henselae DNA and specific antibody prevalence in 171 mongooses from all six parishes in Grenada, West Indies. Almost a third (32.3%, 54/167) of the tested mongooses were B. henselae seropositive and extracted DNA from 18/51 (35.3%) blood pellets were PCR positive for the citrate synthase (gltA) and/or the ß subunit of RNA polymerase (rpoB) genes. All sequences were identical to B. henselae genotype I, as previously reported from Japan. This study confirms the role of small Indian mongooses as a natural reservoir of B. henselae in the New World.