Expansion spatial transcriptomics.

Capture array-based spatial transcriptomics methods have been widely used to resolve gene expression in tissues; however, their spatial resolution is limited by the density of the array. Here we present expansion spatial transcriptomics to overcome this limitation by clearing and expanding tissue prior to capturing the entire polyadenylated transcriptome with an enhanced protocol. This approach enables us to achieve higher spatial resolution while retaining high library quality, which we demonstrate using mouse brain samples.

Protocol to detect RNAs from tissue sections in mice using Y-branched probe in situ hybridization.

Here, we describe a fluorescent in situ hybridization protocol named Yn-situ, standing for Y-branched probe in situ hybridization, to detect RNAs from mice tissue sections. We provide steps for the synthesis and quantification of preamplifier probe using nickase. We also detail the preparation of tissue section, probe hybridization, signal development using hybridization chain reaction (HCR), and quantification of the signals. This approach avoids the use of proprietary in situ hybridization techniques, therefore reducing costs. For complete details on the use and execution of this protocol, please refer to Wu et al. (2022).

TGS1 impacts snRNA 3'-end processing, ameliorates survival motor neuron-dependent neurological phenotypes in vivo and prevents neurodegeneration.

Trimethylguanosine synthase 1 (TGS1) is a highly conserved enzyme that converts the 5'-monomethylguanosine cap of small nuclear RNAs (snRNAs) to a trimethylguanosine cap. Here, we show that loss of TGS1 in Caenorhabditis elegans, Drosophila melanogaster and Danio rerio results in neurological phenotypes similar to those caused by survival motor neuron (SMN) deficiency. Importantly, expression of human TGS1 ameliorates the SMN-dependent neurological phenotypes in both flies and worms, revealing that TGS1 can partly counteract the effects of SMN deficiency. TGS1 loss in HeLa cells leads to the accumulation of immature U2 and U4atac snRNAs with long 3' tails that are often uridylated. snRNAs with defective 3' terminations also accumulate in Drosophila Tgs1 mutants. Consistent with defective snRNA maturation, TGS1 and SMN mutant cells also exhibit partially overlapping transcriptome alterations that include aberrantly spliced and readthrough transcripts. Together, these results identify a neuroprotective function for TGS1 and reinforce the view that defective snRNA maturation affects neuronal viability and function.

Robust and sensitive in situ RNA detection using Yn-situ.

We describe a cost-effective, highly sensitive, and quantitative method for in situ detection of RNA molecules in tissue sections. This method, dubbed Yn-situ, standing for Y-branched probe in situ hybridization, uses a single-strand DNA preamplifier with multiple initiation sites that trigger a hybridization chain reaction (HCR) to detect polynucleotides. By characterizing the performance of this method, we show that the Yn-situ method, in conjunction with an improved fixation step, is sensitive enough to allow detection of RNA molecules using fewer probes targeting short nucleotide sequences than existing methods. A set of five probes can produce quantitative results with smaller puncta and higher signal-to-noise ratio than the 20-probe sets commonly required for HCR and RNAscope. We show that the high sensitivity and wide dynamic range allow quantification of genes expressed at different levels in the olfactory sensory neurons. We describe key steps of this method to enable broad utility by individual laboratories.

The complete mitogenome of giant freshwater prawn (Macrobrachium rosenbergii) from two different selective breeding populations in China.

The giant freshwater prawn (GFP), Macrobrachium rosenbergii, is one of the largest freshwater shrimps in the world, being widely cultured because of its high economic value. In this study, complete mitogenomes of two GFP individuals from different selective breeding populations, 'South Taihu No.2' (ST) and 'Shufeng' (SF), were newly sequenced, compared with each other, and with those of other published Macrobrachium species. The total length is 15,767 bp (ST) and 15,766 bp (SF), including 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and one control region. The phylogenetic analyses based on whole mitogenome sequences suggest that 'Shufeng' has a slightly distant relationship with 'South Taihu No.2', with a pairwise genetic distance of 0.011. This study can provide a genetic background for the GFP selective breeding, and add significantly to the knowledgebase regarding crustacean biology and aquaculture as well.

Encoding innately recognized odors via a generalized population code.

Odors carrying intrinsic values often trigger instinctive aversive or attractive responses. It is not known how innate valence is encoded. An intuitive model suggests that the information is conveyed through specific channels in hardwired circuits along the olfactory pathway, insulated from influences of other odors, to trigger innate responses. Here, we show that in mice, mixing innately aversive or attractive odors with a neutral odor and, surprisingly, mixing two odors with the same valence, abolish the innate behavioral responses. Recordings from the olfactory bulb indicate that odors are not masked at the level of peripheral activation and glomeruli independently encode components in the mixture. In contrast, crosstalk among the mitral and tufted (M/T) cells changes their patterns of activity such that those elicited by the mixtures can no longer be linearly decoded as separate components. The changes in behavioral and M/T cell responses are associated with reduced activation of brain areas linked to odor preferences. Thus, crosstalk among odor channels at the earliest processing stage in the olfactory pathway leads to re-coding of odor identity to abolish valence associated with the odors. These results are inconsistent with insulated labeled lines and support a model of a common mechanism of odor recognition for both innate and learned valence associations.

Acquisition of innate odor preference depends on spontaneous and experiential activities during critical period.

Animals possess an inborn ability to recognize certain odors to avoid predators, seek food, and find mates. Innate odor preference is thought to be genetically hardwired. Here we report that acquisition of innate odor recognition requires spontaneous neural activity and is influenced by sensory experience during early postnatal development. Genetic silencing of mouse olfactory sensory neurons during the critical period has little impact on odor sensitivity, discrimination, and recognition later in life. However, it abolishes innate odor preference and alters the patterns of activation in brain centers. Exposure to innately recognized odors during the critical period abolishes the associated valence in adulthood in an odor-specific manner. The changes are associated with broadened projection of olfactory sensory neurons and expression of axon guidance molecules. Thus, a delicate balance of neural activity is needed during the critical period in establishing innate odor preference and convergent axon input is required to encode innate odor valence.

A Population of Navigator Neurons Is Essential for Olfactory Map Formation during the Critical Period.

In the developing brain, heightened plasticity during the critical period enables the proper formation of neural circuits. Here, we identify the "navigator" neurons, a group of perinatally born olfactory sensory neurons, as playing an essential role in establishing the olfactory map during the critical period. The navigator axons project circuitously in the olfactory bulb and traverse multiple glomeruli before terminating in perspective glomeruli. These neurons undergo a phase of exuberant axon growth and exhibit a shortened lifespan. Single-cell transcriptome analyses reveal distinct molecular signatures for the navigators. Extending their lifespan prolongs the period of exuberant growth and perturbs axon convergence. Conversely, a genetic ablation experiment indicates that, despite postnatal neurogenesis, only the navigators are endowed with the ability to establish a convergent map. The presence and the proper removal of the navigator neurons are both required to establish tight axon convergence into the glomeruli.

Regeneration and rewiring of rodent olfactory sensory neurons.

The olfactory sensory neurons are the only neurons in the mammalian nervous system that not only regenerate naturally and in response to injury, but also project to specific targets in the brain. The stem cells in the olfactory epithelium commit to both neuronal and non-neuronal lineages depending on the environmental conditions. They provide a continuous supply of new neurons. A newly generated neuron must express a specific odorant receptor gene and project to a central target consist of axons expressing the same receptor type. Recent studies have provided insights into this highly regulated, complex process. However, the molecular mechanisms that determine the regenerative capacity of stem cells, and the ability of newly generated neurons in directing their axons toward specific targets, remain elusive. Here we review progresses and controversies in the field and offer testable models.

A developmental switch of axon targeting in the continuously regenerating mouse olfactory system.

The mammalian olfactory system has the natural capacity to regenerate throughout the animal's life span. Despite constant neurogenesis, olfactory sensory neurons project to precise, stereotypical positions in the brain. Here, we identify a critical period of olfactory sensory axon targeting during postnatal development in mouse. Perturbing axon projection beyond postnatal day 7 permanently disrupts targeting specificity of the sensory neurons. In addition, we find that the establishment of the convergence map requires perinatal sensory neurons. Late-born neurons appear to connect with prospective glomeruli based on homotypic interactions among neurons expressing the same odorant receptor. Our results reveal a developmental switch in axon guidance and a mechanism of circuit integration of adult-born neurons.

[LLE-PLS nonlinear modeling method for near infrared spectroscopy and its application].

The traditional near infrared (NIR) spectra modeling algorithm-partial least squares (PLS) can't effectively reflect the nonlinear correlations existing between the near infrared spectra and the chemical or physical properties of samples. Locally linear embedding (LLE) is a newly proposed nonlinear dimension reduction algorithm, which is a kind of manifold learning algorithm. It can find out the intrinsic dimension from high dimensional data effectively, and map the high dimensional input data points to a global low dimensional coordinates while keeping the spatial relations of the adjacent points, i. e. the geometry structure of the high dimensional space. No application of LLE in the information processing of NIR spectra has been reported. By combining LLE and PLS, a novel nonlinear modeling method LLE-PLS for NIR spectra was proposed. In the proposed method, LLE and PLS were adopted to deduct the dimensions of NIR spectra and build regressor, respectively. The LLE-PLS method was applied to correlate the NIR spectra with the concentrations of salvia acid B in the elution of column chromatography of Salvianolate. The results showed that LLE-PLS outperformed other preprocessing methods such as multiplicative scattering correction, the 1st derivative, vector normalization, minimum-maximum normalization, detrend, debias, and the 2nd derivative. After parameter optimization, LLE-PLS can accurately predict the concentration of salvia acid B, with a minimum RMSECV of 0.128 mg x mL(-1) and r2 of 0.9988, suggesting that LLE-PLS is better than PLS in modeling and prediction. The parameter of the number of nearest neighbor k of LLE-PLS and output dimension d can affect the performance of the method. The research showed that k is robust to RMSECV, and an excessively low or high output dimension d will result in a greater error because of insufficient or excessive information extraction. It can be concluded that LLE-PLS can effectively model the nonlinear correlations between spectra and physicochemical properties of the samples. And it is feasible to actualize online monitoring of the process of column chromatography of Salvianolate by coupling NIR spectra with LLE-PLS modeling method.

Ginkgo biloba extract suppresses hypertrophy and extracellular matrix accumulation in rat mesangial cells.

AIM: To observe the effects of Ginkgo biloba extract (EGb) on the hypertrophy of mesangial cells and the accumulation of extracellular matrix (ECM) in mesangial cells. METHODS: Cultured mesangial cells were allotted into 7 groups: normal group, solvent control group, high glucose group, low dose of EGb group, moderate dose of EGb group, high dose of EGb group, and captopril group. Activities of cell antioxidases, S phase percentage and G(0)/G(1) phase percentage, collagen IV and laminin, Smad2/3 and Smad7, TGF-beta(1) mRNA were measured by different methods. RESULTS: For EGb-treated groups, when compared with high glucose group, the cell percentage of S phase was raised and the percentage of G(0)/G(1) was lowered. The intensity of oxidative stress was weakened. The expression of Smad2/3 was greatly decreased and Smad7 was increased. Collagen IV, laminin and TGF- beta(1)mRNA were also reduced. CONCLUSION: EGb can suppress cell hypertrophy and the accumulation of ECM in rat mesangial cells, which means it could play a vital role in the delay of glomerulosclerosis in diabetic nephropathy.