Multi-Omic Biomarkers Improve Indeterminate Pulmonary Nodule Malignancy Risk Assessment.

The clinical management of patients with indeterminate pulmonary nodules is associated with unintended harm to patients and better methods are required to more precisely quantify lung cancer risk in this group. Here, we combine multiple noninvasive approaches to more accurately identify lung cancer in indeterminate pulmonary nodules. We analyzed 94 quantitative radiomic imaging features and 41 qualitative semantic imaging variables with molecular biomarkers from blood derived from an antibody-based microarray platform that determines protein, cancer-specific glycan, and autoantibody-antigen complex content with high sensitivity. From these datasets, we created a PSR (plasma, semantic, radiomic) risk prediction model comprising nine blood-based and imaging biomarkers with an area under the receiver operating curve (AUROC) of 0.964 that when tested in a second, independent cohort yielded an AUROC of 0.846. Incorporating known clinical risk factors (age, gender, and smoking pack years) for lung cancer into the PSR model improved the AUROC to 0.897 in the second cohort and was more accurate than a well-characterized clinical risk prediction model (AUROC = 0.802). Our findings support the use of a multi-omics approach to guide the clinical management of indeterminate pulmonary nodules.

Transcriptomic analyses of NeuroD1-mediated astrocyte-to-neuron conversion.

Ectopic expression of a single neural transcription factor NeuroD1 can reprogram reactive glial cells into functional neurons both in vitro and in vivo, but the underlying mechanisms are not well understood yet. Here, we used RNA-sequencing technology to capture the transcriptomic changes at different time points during the reprogramming process. We found that following NeuroD1 overexpression, astroglial genes (ACTG1, ALDH1A3, EMP1, CLDN6, SOX21) were significantly downregulated, whereas neuronal genes (DCX, RBFOX3/NeuN, CUX2, RELN, SNAP25) were significantly upregulated. NeuroD family members (NeuroD1/2/6) and signaling pathways (Wnt, MAPK, cAMP) as well as neurotransmitter receptors (acetylcholine, somatostatin, dopamine) were also significantly upregulated. Gene co-expression analysis identified many central genes among the NeuroD1-interacting network, including CABP7, KIAA1456, SSTR2, GADD45G, LRRTM2, and INSM1. Compared to chemical conversion, we found that NeuroD1 acted as a strong driving force and triggered fast transcriptomic changes during astrocyte-to-neuron conversion process. Together, this study reveals many important downstream targets of NeuroD1 such as HES6, BHLHE22, INSM1, CHRNA1/3, CABP7, and SSTR2, which may play critical roles during the transcriptomic landscape shift from a glial profile to a neuronal profile.

Dynamic Changes in Breast Milk Microbiome in the Early Postpartum Period of Kenyan Women Living with HIV Are Influenced by Antibiotics but Not Antiretrovirals.

Shared bacteria between maternal breast milk and infant stool, infers that transfer of maternal breast milk microbiota through breastfeeding seeds the establishment of the infant gut microbiome. Whether combination antiretroviral therapy (cART) impacts the breast milk microbiota in women living with HIV is unknown. Since current standard of care for people living with HIV includes cART, it has been difficult to evaluate the impact of cART on the microbiome. Here, we performed a next-generation sequencing retrospective study from pre-ART era clinical trials in Nairobi, Kenya (between 2003-2006 before cART was standard of care) that tested the effects of ART regimens to prevent mother-to-child HIV transmission. Kenyan women living with HIV were randomized to receive either no ART during breastfeeding (n = 24) or cART (zidovudine, nevirapine, lamivudine; n = 25) postpartum. Using linear mixed-effects models, we found that alpha diversity and beta diversity of the breast milk bacterial microbiome changed significantly over time during the first 4 weeks postpartum (alpha diversity P < 0.0007; beta diversity P = 0.005). There was no statistically significant difference in diversity, richness, and composition of the bacterial microbiome between cART-exposed and cART-unexposed women. In contrast, antibiotic use influenced the change of beta diversity of the bacterial microbiome over time. Our results indicate that while early postpartum time predicts breast milk microbiome composition, cART does not substantially alter the breast milk microbiota in women living with HIV. Hence, cART has minimal impact on the breast milk microbiome compared to antibiotics use. IMPORTANCE Breastfeeding has important benefits for long-term infant health, particularly in establishing and shaping the infant gut microbiome. However, the impact of combination antiretroviral therapy exposure and antibiotics on the breast milk microbiome in women living with HIV is not known. Here, in a longitudinal retrospective study of Kenyan women living with HIV from the pre-antiretroviral therapy era, we found that antibiotic use significantly influenced breast milk microbiome beta diversity, but antiretrovirals exposure did not substantially alter the microbiome. Given the protective role of breastfeeding in maternal-infant health, these findings fill an important knowledge gap of the impact of combination antiretroviral therapy on the microbiome of women living with HIV.

A NeuroD1 AAV-Based Gene Therapy for Functional Brain Repair after Ischemic Injury through In Vivo Astrocyte-to-Neuron Conversion.

Adult mammalian brains have largely lost neuroregeneration capability except for a few niches. Previous studies have converted glial cells into neurons, but the total number of neurons generated is limited and the therapeutic potential is unclear. Here, we demonstrate that NeuroD1-mediated in situ astrocyte-to-neuron conversion can regenerate a large number of functional new neurons after ischemic injury. Specifically, using NeuroD1 adeno-associated virus (AAV)-based gene therapy, we were able to regenerate one third of the total lost neurons caused by ischemic injury and simultaneously protect another one third of injured neurons, leading to a significant neuronal recovery. RNA sequencing and immunostaining confirmed neuronal recovery after cell conversion at both the mRNA level and protein level. Brain slice recordings found that the astrocyte-converted neurons showed robust action potentials and synaptic responses at 2 months after NeuroD1 expression. Anterograde and retrograde tracing revealed long-range axonal projections from astrocyte-converted neurons to their target regions in a time-dependent manner. Behavioral analyses showed a significant improvement of both motor and cognitive functions after cell conversion. Together, these results demonstrate that in vivo cell conversion technology through NeuroD1-based gene therapy can regenerate a large number of functional new neurons to restore lost neuronal functions after injury.

Transcriptome Analysis of Small Molecule-Mediated Astrocyte-to-Neuron Reprogramming.

Chemical reprogramming of astrocytes into neurons represents a promising approach to regenerate new neurons for brain repair, but the underlying mechanisms driving this trans-differentiation process are not well understood. We have recently identified four small molecules - CHIR99021, DAPT, LDN193189, and SB431542 - that can efficiently reprogram cultured human fetal astrocytes into functional neurons. Here we employ the next generation of RNA-sequencing technology to investigate the transcriptome changes during the astrocyte-to-neuron (AtN) conversion process. We found that the four small molecules can rapidly activate the hedgehog signaling pathway while downregulating many glial genes such as FN1 and MYL9 within 24 h of treatment. Chemical reprogramming is mediated by several waves of differential gene expression, including upregulation of hedgehog, Wnt/ß-catenin, and Notch signaling pathways, together with downregulation of TGF-ß and JAK/STAT signaling pathways. Our gene network analyses reveal many well-connected hub genes such as repulsive guidance molecule A (RGMA), neuronatin (NNAT), neurogenin 2 (NEUROG2), NPTX2, MOXD1, JAG1, and GAP43, which may coordinate the chemical reprogramming process. Together, these findings provide critical insights into the molecular cascades triggered by a combination of small molecules that eventually leads to chemical conversion of astrocytes into neurons.

Chemical Conversion of Human Fetal Astrocytes into Neurons through Modulation of Multiple Signaling Pathways.

We have previously developed a cocktail of nine small molecules to convert human fetal astrocytes into neurons, but a nine-molecule recipe is difficult for clinical applications. Here, we identify a chemical formula with only three to four small molecules for astrocyte-to-neuron conversion. We demonstrate that modulation of three to four signaling pathways among Notch, glycogen synthase kinase 3, transforming growth factor ß, and bone morphogenetic protein pathways is sufficient to change an astrocyte into a neuron. The chemically converted human neurons can survive >7 months in culture, fire repetitive action potentials, and display robust synaptic burst activities. Interestingly, cortical astrocyte-converted neurons are mostly glutamatergic, while midbrain astrocyte-converted neurons can yield some GABAergic neurons in addition to glutamatergic neurons. When administered in vivo through intracranial or intraperitoneal injection, the four-drug combination can significantly increase adult hippocampal neurogenesis. Together, human fetal astrocytes can be chemically converted into functional neurons using three to four small molecules, bringing us one step forward for developing future drug therapy.

Small Molecules Efficiently Reprogram Human Astroglial Cells into Functional Neurons.

We have recently demonstrated that reactive glial cells can be directly reprogrammed into functional neurons by a single neural transcription factor, NeuroD1. Here we report that a combination of small molecules can also reprogram human astrocytes in culture into fully functional neurons. We demonstrate that sequential exposure of human astrocytes to a cocktail of nine small molecules that inhibit glial but activate neuronal signaling pathways can successfully reprogram astrocytes into neurons in 8-10 days. This chemical reprogramming is mediated through epigenetic regulation and involves transcriptional activation of NEUROD1 and NEUROGENIN2. The human astrocyte-converted neurons can survive for >5 months in culture and form functional synaptic networks with synchronous burst activities. The chemically reprogrammed human neurons can also survive for >1 month in the mouse brain in vivo and integrate into local circuits. Our study opens a new avenue using chemical compounds to reprogram reactive glial cells into functional neurons.