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

9/22
Preparation of Segmented and Polarity Marked Microtubules
1. Solution and supply
1. BRB80 (1X) - (generally made as a 5X stock and stored at 4¡C)
1. 80 mM PIPES
2. 1 mM MgCl2
3. 1 mM EGTA
4. pH 6.8 with KOH
2. 100 mM GTP
3. 100 mM GMPCPP
4. 50 nM NEM
5. Bright GMPCPP Seed Mix (2 mg/ml; 1 part rhodamine tubulin to 2 parts unlabeled tubulin; prepared and stored at -80C as described above)
6. NEM CPP-Tubulin (prepared by treating recycled tubulin (~5-15 mg/ml) in BRB80 + 0.5 mM GMPCPP with 1 mM NEM (freshly prepared as a 50 mM stock in water) for 10' at 0C, quenching NEM with 8 mM ꞵ-mercaptoethanol for 10' at 0C, freezing aliquots in liquid nitrogen and storing at -80C.
2. Polarity Marked GMPCPP Microtubules
1. Prepare dim GMPCPP polar elongation mix: 10 µM (1 mg/ml) 1:9 rhodamine labeled : unlabeled tubulin and 8 µM NEM CPP-Tubulin in 1X BRB80, 1 mM DTT, 0.5 mM GMPCPP. Incubate on ice for 5'-10', spin 90K 5' in TLA100 at 2C, freeze in liquid nitrogen in 10 µl aliquots (or use fresh).
2. Thaw GMPCPP bright seed mix by adding 9 vol of warm (37C) BRB80 + 1 mM DTT (2 µM tubulin final) and incubate at 37C for 30’ - 45’.
3. Thaw CPP polar elongation mix and store on ice. Dilute as follows on ice: 17 µl BRB80 + 1 mM DTT 3 µl CPP Polar Elongation Mix (This results in elongation of ~1.5 µM CPP-tubulin)
4. Incubate diluted CPP polar elongation mix at 37C for 20 sec before adding 2 µl of polymerized bright CPP seeds.
5. Incubate at 37C for 1-2 hours. Pellet and resuspend or use directly.

9/22
Quality assurance of hematopoietic stem cells by macrophages determines stem cell clonality (Science 2022 Havard Leonard I. Zon)
Hematopoietic stem cells have been found to regulate the number of hematopoietic stem cell clones in adulthood by interacting with macrophages to ensure hematopoietic stem cell quality.
Through in vivo high-resolution imaging, the authors found that 30% of hematopoietic stem and progenitor cells interact with macrophages (>45 min), which is much higher than the interaction between macrophages and erythrocytes and endothelial cells - Contact with macrophages may aid in quality control of hematopoietic stem and progenitor cells prior to their large-scale expansion
Using antibody immunostaining, the authors found that calreticulin forms punctate signals on the surface of hematopoietic stem and progenitor cells and promotes interaction with macrophages

9/24
Reconstitution of the tubular endoplasmic reticulum network with purified components (Nature 2017 Harvard Tom Rapoport)
reconstitute a dynamic tubular membrane network with purified endoplasmic reticulum proteins
Sey1p: the membrane-fusing GTPase
Yop1p: the curvature-stabilizing protein
Sey1p + Yop1p: form a tubular network upon addition of GTP
Inhibit Sey1p: the tubules rapidly fragment - network maintenance requires continuous membrane fusion
Yop1p favours the generation of highly curved membrane structures
Their results show that organelle shape can be generated by a surprisingly small set of proteins and represents an energy-dependent steady state between formation and disassembly.

9/27
Timing of daily calorie loading affects appetite and hunger responses without changes in energy metabolism in healthy subjects with obesity (2022 Cell Metabolism)
The experimental results clearly demonstrate that calorie utilization does not vary with time of day, contrary to most existing studies
Changes in weight loss seen in previous studies from eating more breakfast may be behavioral, such as changes in appetite

9/27
DNA replication timing directly regulates the frequency of oncogenic chromosomal translocations (Science 2022 IMP Rushad Pavri)
Chromosomal translocations, arising from inappropriate junctions that occur after DNA double-strand breaks, involving proto-oncogenes are a common feature of cancer
After chromosomal translocation of proto-oncogene, dysregulation of proto-oncogene expression leads to tumorigenesis
The study raises the idea that replication clusters of distinct chromosomes involved in two distinct functions, such as activation of DNA replication origins and DNA repair functions, could interact in a common hub, and that interchromosomal interactions could lead to genetic instability Abnormal reorganization, further leading to tumorigenesis
During antibody maturation in B cells, DNA replication timing (RT) directly regulates the occurrence of MYC-IGH gene translocation in lymphoma, and chromatin folding results in the spatial proximity of MYC and IGH, resulting in a replication center. Origins of replication are activated synchronously, facilitating this translocation. RT forms the basis between DNA double-strand break DSB formation and DSB junctions during chromosomal translocations

9/27
An intercellular transfer of telomeres rescues T cells from senescence and promotes long-term immunological memory (Nature Cell Biology 2022)
Telomeres are repeating sequences composed of TTAGGG that protect the ends of chromosomes and affect the lifespan of cells.
Telomeres shorten as cells divide, eventually leading to cell senescence.
Telomerase can help prolong the length of telomeres.
Although T cells can use telomerase to alleviate the shortening of telomeres caused by their rapid clonal expansion, the activation of telomerase is not enough to prevent the exhaustion of T cells, and ultimately still senescent T cells
Immune synapses are antigen-specific junctions formed between antigen-presenting cells (APCs) and lymphocytes, which trigger immune protective responses and ultimately generate long-lived memory T cells.
Synaptic stimulation leads to telomerase activation in T cells, whereas repeated immune synaptic interactions lead to a gradual decline in T cell telomerase activation, manifesting as T cell senescence
The article found that after some T cells can lengthen their own telomeres by acquiring telomeres from extracellular vesicles secreted by antigen-presenting cells (APCs), these T cells acquire telomeres and become long-lived centers similar to stem cells. Memory cells, which maintain long-term immune memory, while other T cells gradually senesce.

9/29
Microbiota imbalance induced by dietary sugar disrupts immune-mediated protection from metabolic syndrome (Cell Columbia 2022 Ivaylo I. Ivanov)
This study found that Th17-inducing bacteria can prevent or treat metabolic syndrome and type 2 diabetes by regulating lipid absorption in the small intestine
Sugar in dietary components leads to lower abundance of SFB and lower proportion of Th17 cells, but not fat content or fiber

9/29
mTOR-regulated mitochondrial metabolism limits mycobacterium-induced cytotoxicity (Cell 2022 Lalita Ramakrishnan Cambridge)
Mycobacterium tuberculosis (Mtb) spreads through the air and enters the lungs of patients, forming granulomas containing immune cells such as Mycobacterium tuberculosis and macrophages.
Using the zebrafish experimental system, the researchers discovered that mTOR kinase is one of the pioneers in the body's resistance to tuberculosis infection. mTOR can regulate mitochondrial metabolism to prevent macrophage death and gain precious time for immunity.

9/29
Independent origins of fetal liver haematopoietic stem and progenitor cells (Nature 2022 Toshio Suda Japan)
Homeostasis of Haematopoietic stem cells (HSCs) in adult bone marrow depends on self-renewal and tight control of differentiation
It is unclear how the seemingly opposing tasks of hematopoietic stem cell self-renewal and differentiation are accomplished during the brief period of embryonic development
The article analyzes the origin of hematopoietic stem and progenitor cells in the arteries by using genetic tracing technology in mice
Hematopoietic stem or progenitor cells are transformed from endothelial cells through the process of Endothelialto-haematopoietic transition (EHT)
Hematopoietic stem/progenitor precursor cells in fetal liver are generated independently of hematopoietic stem cells in bone marrow
The expression level of EVI1 is important for hematopoietic stem/progenitor precursor cell fate determination in vivo, and this finding can be used to guide the induction of hematopoietic stem cells in vitro.