9/6
Microtubule dynamics (JCS 2002 Rebecca Heald)
Microtubules are highly dynamic and switch stochastically between growing and shrinking phases both in vivo and in vitro.
The GTP-cap model proposes that the body of the microtubule, which comprises GDP-tubulin subunits, is unstable. The microtubule structure is stabilized by a layer of GTP tubulin subunits at the end that may act to maintain association between protofilaments. When this cap is stochastically lost, the protofilaments peel outward and the microtubule rapidly depolymerizes.
Stabilize/ destabilize
microtubule-organizing centers/MTOC: composed of a pair of cylindrical centrioles surrounded by pericentriolar material containing an isoform of tubulin (γ-tubulin) in a large complex that includes other proteins (collectively known as ‘grips’) and functions as a nucleating seed: the γ-tubulin ring complex (γ-TuRC)

Stability: microtubule-associated proteins (MAPs)
bind to the surface of the microtubule
bridging several tubulin subunits
possibly neutralizing the repulsive negative charge on the microtubule surface
Distability
The microtubule-destabilizing factor katanin functions as a severing factor, generating new ends lacking a GTP cap
Depolymerizing kinesins of the KinI family bind to microtubule ends and distort the microtubule lattice, forcing protofilament peeling

9/7
Design, construction, and in vivo augmentation of a complex gut microbiome (Cell 2022 Michael A. Fischbach Kerwyn Casey Huang Stanford University)
A well-defined, highly complex (containing 119 strains) synthetic microbial community that more effectively reflects the role of the human gut microbiota was constructed.
Sequencing by metagenomic sequencing and developed a new algorithm - NinjaMap
Bowtie2, SAMtools: high sensitivity but low accuracy
Kraken2, MetaPhlAn2: low sensitivity
hCom1 - construct and characterize in vitro a defined community of 104 bacterial species
hCom2 - identified new species that engrafted following fecal challenge and added them to hCom1

9/7
Purified kinesin promotes vesicle motility and induces active sliding between microtubules in vitro (PNAS 1991 JHU)
Kinesin induced the formation of microtubule aster with the plus ends of microtubules located at the center of each aster
Formed small foci first and then the foci coalesce into large asters

9/8
Translatome and transcriptome co-profiling reveals a role of TPRXs in human zygotic genome activation (2022 Science 清华 Wei xie)
oocyte-to-embryo transition (OET)
human zygotic genome activation (ZGA): The first transcriptional events at the onset of life and key events that initiate embryonic developmental processes - still unclear
This paper made the translational map during the transition from human eggs to early embryos, revealing the differences in the dynamic changes of translation levels in human-mouse eggs and early embryos
Identifies a group of Homeobox transcription factors, including TPRXL, TPRX1, and TPRX2, as key regulators of human zygotic genome activation
Method - R2-lite (​​Ribo-RNA-lite): a combination of Ribo-lite (Ligation-free, ultra-low-InpuT and Enhanced Ribo-seq) - Ultrasensitive translational genome sequencing technology and Smart-seq2 - Transcriptome Sequencing Technology.
Transcriptome: A transcriptome is the full range of messenger RNA, or mRNA, molecules expressed by an organism
Translational genome: DNA set

9/8
High-throughput continuous evolution of compact Cas9 variants targeting single-nucleotide-pyrimidine PAMs (Nature biotechnology 2022 David Liu; Ahmad S. Khalil - BU)
In recent years, researchers have used various strategies to upgrade SpCas9, which has expanded its PAM recognition sequence from traditional NGG to NG, NRN and even NYN, effectively expanding the application prospects of SpCas9 and its derivatives
Mutants of SpCas9 are well-edited at purine-like PAM sites (NRN), but still insufficient at pyrimidine-like PAM sites (NYN).
SpCas9 also has shortcomings such as its large size (1368 amino acids), which is difficult for it to deliver.
With the help of the upgraded protein-directed evolution system, a series of optimization and transformation of the small Cas9 system Nme2Cas9 was carried out. The modified Nme2Cas9 mutant successfully broke through the limitation of traditional PAM (N4CC), which can effectively identify N4YN-type PAM and achieve efficient gene editing.

9/9
Excitatory SST neurons in the medial paralemniscal nucleus control repetitive self-grooming and encode reward (Neuron 2022 上交 Ju Huang)
Excitatory SST neurons in the medial parabrachial nucleus control repetitive self-grooming and encode reward
Human body-focused repetitive behaviors (BFRBs) refer to a set of periodic non-functional behaviors directed at the body (such as hair pulling and skin scratching or intense urges like biting, picking, and pulling that can cause damage)
BFRBs may have emotion-regulating effects, allowing individuals to manage negative emotions under stressful exposure by reducing arousal and resisting anxiety

9/12
Geometric trade-off between contractile force and viscous drag determines the actomyosin-based motility of a cell-sized droplet (2022 PNAS Kyushu University)
propulsive force can be generated by the physical interaction between contracting active cytoskeleton and inner droplet surface
the force balance between propulsive force and confinement-induced viscous drag determines the migration speed, revealing a physical mechanism of the active cytoskeleton-based motility that utilizes environmental mechanical constraints
A new in vitro migratory cell model: cytoplasmic actomyosin networks are encapsulated into droplets surrounded by a lipid monolayer membrane

9/12
Structural basis of nucleosome disassembly and reassembly by RNAPII elongation complex with FACT (Science Japan 2022)
Within the nucleus of eukaryotic cells, genomic DNA forms a highly ordered compact structure called chromatin. The basic unit of chromatin is the nucleosome, which is formed by 147bp DNA wrapped around a histone octamer, which contains two copies of each of the four histones (H2A, H2B, H3, H4).
The researchers used cryo-electron microscopy to resolve multiple structures of the RNAPII extension complex (EC) through the nucleosome, captured the details of the EC's advancement on DNA, and found that EC can mediate downstream nucleosome depolymerization and upstream reassembly, The histone chaperone FACT facilitates this process

9/12
Immune-microbe interactions early in life: A determinant of health and disease long term (Science 2022 Petter Brodin Imperial College London)
This article explores the interactions between the microbiome and the host immune system around the prenatal, perinatal and postnatal phases, and discusses early microbiome perturbations and mismatches between "ancestral" and modern environments effects on health and disease.
Prenatal phase - The role of transplacentally transferred microbial components may be to provide antigens to fetal T cells, thereby lowering their activation threshold in preparation for subsequent microbial colonization and preventing an excessive immune response.
Perinatal phase - Different modes of delivery lead to large differences in the microbiome in the early postpartum period - However, microbes acquired through vaginal delivery and those from the gut are better suited for surviving in the baby's gut
Postnatal phase - After birth, immune cells must be able to recognize the microbes that colonize the body and make the right decision—accept them or kill them.

9/14
The TRESLIN-MTBP complex couples completion of DNA replication with S/G2 transition (2022 Molecular Biology Luis Toledo University of Copenhagen)
Cell cycle checkpoints in yeast are controlled by Mec1 and Rad53 kinases
Homologs of Mec1 and Rad53 in humans are ATR and CHK1, respectively, and were found to be key responders to replication stress (RS) and DNA damage
ATR/CHK1: Under normal conditions, they are also active in S phase, and throughout S phase cells rely on this ATR/CHK1 activity to limit CDK2 activity and regulate the triggering of origins of replication - DNA Replication Monitoring System
However, the ability of ATR/CHK1 to prevent premature entry into mitosis is rather limited

This article finds that the TRESLIN-MTBP complex monitors the triggering of origins of replication to couple DNA replication to the timely transition from S phase to G2 phase, thereby revealing a protective mechanism independent of the canonical ATR/CHK1 pathway in mammals. A key function of cell cycle regulation in early entry into G2 phase from early S phase.
The TRESLIN-MTBP complex possesses the controlled ability to simultaneously promote DNA replication and inhibit entry into G2 phase

9/14
Living material assembly of bacteriogenic protocells (Nature 2022 University of Bristol Stephen Mann)
Protocells: Artificial construction with functions similar to living cells
The researchers broke the traditional method of using non-living substances to construct artificial cells, developed a method for spatially controllable assembly and in situ lysis of bacterial colonies using polymer-nucleoside triphosphate condensate droplets, and constructed the first method based on Eukaryotic cell mimicking prokaryotic cell based on prokaryotic cell
The primary cell inherits about 85% of the bioactive components in the live bacterial cells after in situ lysis, and exhibits living cell-like characteristics including gene expression, glycolysis, and enzyme catalysis.
This cell system includes a spatially partitioned DNA–histone nucleus-like condensate, membranized water vacuoles and a three-dimensional network of F-actin proto-cytoskeletal filaments.

9/16
Combined alcohol and cannabinoid exposure leads to synergistic toxicity by affecting cerebellar Purkinje cells (Nature Metabolism 2022 中科大 Wei Xiong)
Combined use of cannabis and alcohol (Ethanol + Δ9-tetrahydrocannabinol (THC)) results in greater psychoactive toxicity than either substance alone
This study reveals the neural mechanism by which alcohol and cannabinoids target the presynaptic cannabinoid receptor (CB1R) and extrasynaptic glycine receptor (esGlyR) in cerebellar Purkinje cells to synergistically lead to ataxia, and propose the development of targeted esGlyR's novel drug is used to alleviate this movement disorder, further accelerating the clinical treatment of alcohol and cannabis abuse.

9/20
Pilot study of responsive nucleus accumbens deep brain stimulation for loss-of-control eating (Nature medicine 2022 Casey H. Halpern)
Binge eating disorder (BED) is one of the common eating disorders that can lead to obesity, reduced quality of life, and a shorter lifespan
The results of related animal models show that the electrophysiological activity of the nucleus accumbens is clearly associated with binge-eating-related behavior, and brief high-frequency electrical stimulation can significantly reduce binge-eating behavior in mice
Deep brain stimulation targeting the nucleus accumbens was found to improve food intake and body weight in 2 enrolled severely obese volunteers with binge eating disorder

9/20
(R) Emerging roles and functional mechanisms of PIWI-interacting RNAs (Nature Reviews Molecular Cell Biology 2022)
PIWI-interacting RNA (pi RNA): a new class of small non-coding RNAs that can specifically interact with PIWI family proteins
Compared with microRNAs (miRNAs) and small interfering RNAs (siRNAs), piRNAs are longer, with a length of between 30 nt and 2'-O-methylation at the 3' end.
In terms of processing pathways, miRNA and siRNA are generated by RNase III (Dicer) cleavage of hairpin-shaped or double-stranded precursor transcripts, respectively; while piRNAs are generated from long single-stranded transcripts, and their processing and maturation is independent of Dicer.
PIWI-piRNA complexes are more than just silent transposable elements (TE) but also involved in the regulation of protein-coding gene expression in germ cells
This article summarizes piRNA cluster transcription, piRNA generation, and piRNA pathway mechanisms and functions, and outlines novel roles for piRNAs in Drosophila and mouse germ cell development and human disease.

9/21
Modeling human extraembryonic mesoderm cells using naive pluripotent stem cells (Cell Stem Cell 2022 KU Leuven)
This paper uses primitive human pluripotent stem cells to build a model that mimics the early development of human extraembryonic mesoderm
Extraembryonic mesoderm (EXM): an important tissue in the development process, which is involved in primitive erythropoiesis and extracellular matrix formation.
EXM is closely related to ectoderm, primitive endoderm (PrE) and trophoblast (TB)

9/22
Preparation of Modified Tubulins
1. Cyclin tublin
1. Buffers: Glycerol PB: 80 mM K-PIPES, 5 mM MgC12, l mM EGTA, 1 mM GTP, 33% (v/v) glycerol, pH 6.8
2. BRB80 (The now traditional name for this buffer is derived from Brinkley reassembly buffer)
1. 80 mM K-PIPES
2. 1 mM MgCl2
3. 1 mM EGTA
4. pH 6.8.
2. Biotin-Labeled Tubulin
3. Fluorochrome-Labeled Tubulin
4. N-Ethylmaleimide-Labeled Tubulin - minus-end poly- merization is inhibited > 90%, while plus-end polymerization is inhibited < 10%
1. Thaw cycled tubulin (which is in BRB80) and cool to 0 °. Add GTP to 0.1 mM and then N-ethylmaleimide (from a fresh stock of 50 mM in water) to 1 mM. Incubate at 0° for 10 min. The minimal amount of NEM required is 2 mol/mol tubulin dimer.
2. Add 2-mercaptoethanol to 8 mM. Incubate at 0° for 10 min to inactivate excess NEM. Freeze in aliquots.
5. GTP Analog Seeds
1. analogs, guanylyl (a,ꞵ)-methylene diphos- phonate (GMPCPP) - more stable than GTP microtubules
6. GMPCPP
1. 25 uM cycledtubulin and 500 uM GMPCPP are placed at 37° for 10 min


4 mg/ml rhodamine tubulin is polymerized with 150 pM GMPCPP in BRB80 to 37°C for 15 minutes

9/22
Preparation of Marked Microtubules for the Assay of the Polarity of Microtubule-Based Motors by Fluorescence Microscopy
1. Reagent
1. BRB80 - The buffer used for microtubule polymerization
1. 80 mM 1,4-piperazinediethanesulfonicacid (Pipes)
2. 1 mM GTP
3. 1 mM MgC12
4. pH 6.8
5. with KOH
2. Preparation of the Bright Microtubule Seeds
1. the rhodamine-tubulin should be diluted 1: 1 with unlabeled tubulin.
2. assembly mixtures can be stored as frozen aliquots at -70°C and warmed at 37°C for polymerization
3. Before freezing - all the components are mixed on ice and spun at 30 psi in the airfuge at 4°C for 5 minutes to remove any aggregates
4. The supernatant is fast-frozen in 5 ul aliquots in liquid nitrogen and stored at -70°C.
5. When required, the aliquot can be placed at 37°C to polymerize.
6. Three ways:
1. Stable seeds can be made by polymerizing tubulin with a nonhydrolyzable analog of GTP, GMPCPP - 4 mg/ml rhodamine tubulin is polymerized with 150 uM GMPCPP in BRB80 to 37°C for 15 minutes
2. Short GTP-microtubules can be formed by polymerizing tubulin in the presence of glycerol. Rhodamine-labeled tubulin (4 mg/ml) is placed in BRB8O augmented with 4 mM MgC12and glycerol to 40% (v/v) at 37°C for 30 minutes.
3. large quantities of seeds and can be kept at room temperature for up to 2 months
1. To make glycerol seeds, to 100 ul tubulin 4 mg/ml, add 50 ul glycerol, 1.5 ul 100 mM GTP, and 0.5 ul 1 M MgCI2. Incubate at 37°C for 50 minutes. Shear five times with a 22- to 30-gauge needle.
2. Dilute the glycerol seeds into rhodamine-tubulin at 20 uM. On ice, mix 100 ul tubulin 4 mg/ml, 10 ul rhodamine-tubulin 40 mg/ml, 90 ul BRB80, and 2 uI 100 mM GTP. Warm to 37°C for 1 minute; then add 1/100th volume of seeds. Incubate at 37°C for 60 minutes.
3. Crosslink by adding 1/10th volume of 15 mM EGS (Pierce 21565) in di- methylsulfoxide (DMSO)l). Incubate at 37°C for 60 minutes.
4. Quench by adding 2ml BRB80 containing 50% sucrose, 10 mM potassium glutamate, and 0.1% ꞵ-mercaptoethanol. Incubate at 37°C for 60 minutes.
5. Shear four times with a 22- to 30-gauge needle.
6. Clean the seeds by diluting the seed mixture 1:1 with BRB80 and layering, on a step gradient, 2 ml seeds, 1 ml 40% sucrose in BRB80 plus 0.1% ꞵ-mercaptoethanol, and 2 ml 75% sucrose in BRB80 plus 0.1% ꞵ- mercaptoethanol. Spin 40 minutes 40,000 rpm in a SW 50.1 rotor. Collect seeds at the 40%/75% interface.
3. Preparation of the Polarity-Marked Microtubules
1. The highly labeled seeds are then diluted into a mixture containing a 10:1 molar ratio of unlabeled to rhodamine-labeled tubulin.
2. N- ethylmaleimide (NEM)-treated tubulin - prevents minus-end growth
3. Protocol
1. Prepare a dimly labeled assembly mixture containing unlabeled tubulin at 1.5 mg/ml and rhodamine-tubulin at 0.15 mg/ml with 1 mM GTP in BRB80. Warm to 37°C for 1 minute.
2. After 1 minute, dilute the rhodamine seeds into the tubulin aliquot at a 1:10 (v/v) ratio into the prewarmed assembly mixture. Allow the microtubules to polymerize until they reach a desired length, generally about 10 um.
3. Stabilize the microtubules by adding BRB80 augmented with 10 uM taxol prewarmed to 37°C, taking care to avoid shear.
4. To remove any remaining free rhodamine-tubulin monomer, pellet the taxol-stabilized microtubules in the airfuge at 20 psi for 3 minutes and resuspend in BRB80 augmented with 10 uM taxol.
4. Visualization of Marked Microtubules in Motor Polarity Assays
1. Bleaching - to prevent photobleaching, we use the oxygen-scavenging system developed for the observation of single actin filaments

2. In the motility buffer to be used, 0.1 mg/ml catalase, 0.03 mg/ml glucose oxidase, 10mM glucose, and 0.1% ꞵ-mercaptoethanol are added. With this system, microtubules have been successfully recorded after 10 minutes of illumination with an unattenuated 100-W mercury arc lamp. Microtubule breakage is very rare. Nevertheless, for extended recording, we recommend the use of shuttered light source and a sensitive camera such as a silicon-intensified-target camera