A day in the life of the spliceosome.

One of the most amazing findings in molecular biology was the discovery that eukaryotic genes are discontinuous, with coding DNA being interrupted by stretches of non-coding sequence. The subsequent realization that the intervening regions are removed from pre-mRNA transcripts via the activity of a common set of small nuclear RNAs (snRNAs), which assemble together with associated proteins into a complex known as the spliceosome, was equally surprising. How do cells coordinate the assembly of this molecular machine? And how does the spliceosome accurately recognize exons and introns to carry out the splicing reaction? Insights into these questions have been gained by studying the life cycle of spliceosomal snRNAs from their transcription, nuclear export and re-import to their dynamic assembly into the spliceosome. This assembly process can also affect the regulation of alternative splicing and has implications for human disease.

PIF1 helicase promotes break-induced replication in mammalian cells.

Break-induced replication (BIR) is a specialized homologous-recombination pathway for DNA double-strand break (DSB) repair, which often induces genome instability. In this study, we establish EGFP-based recombination reporters to systematically study BIR in mammalian cells and demonstrate an important role of human PIF1 helicase in promoting BIR. We show that at endonuclease cleavage sites, PIF1-dependent BIR is used for homology-initiated recombination requiring long track DNA synthesis, but not short track gene conversion (STGC). We also show that structure formation-prone AT-rich DNA sequences derived from common fragile sites (CFS-ATs) induce BIR upon replication stress and oncogenic stress, and PCNA-dependent loading of PIF1 onto collapsed/broken forks is critical for BIR activation. At broken replication forks, even STGC-mediated repair of double-ended DSBs depends on POLD3 and PIF1, revealing an unexpected mechanism of BIR activation upon replication stress that differs from the conventional BIR activation model requiring DSB end sensing at endonuclease-generated breaks. Furthermore, loss of PIF1 is synthetically lethal with loss of FANCM, which is involved in protecting CFS-ATs. The breast cancer-associated PIF1 mutant L319P is defective in BIR, suggesting a direct link of BIR to oncogenic processes.

Rapid multiple protein sequence search by parallel and heterogeneous computation.

MOTIVATION: Protein sequence database search and multiple sequence alignment generation is a fundamental task in many bioinformatics analyses. As the data volume of sequences continues to grow rapidly, there is an increasing need for efficient and scalable multiple sequence query algorithms for super-large databases without expensive time and computational costs. RESULTS: We introduce Chorus, a novel protein sequence query system that leverages parallel model and heterogeneous computation architecture to enable users to query thousands of protein sequences concurrently against large protein databases on a desktop workstation. Chorus achieves over 100× speedup over BLASTP without sacrificing sensitivity. We demonstrate the utility of Chorus through a case study of analyzing a ∼1.5-TB large-scale metagenomic datasets for novel CRISPR-Cas protein discovery within 30 min. AVAILABILITY AND IMPLEMENTATION: Chorus is open-source and its code repository is available at https://github.com/Bio-Acc/Chorus.

Circular RNA vaccines against monkeypox virus provide potent protection against vaccinia virus infection in mice.

Since the outbreak of monkeypox (mpox) in 2022, widespread concern has been placed on imposing an urgent demand for specific vaccines that offer safer and more effective protection. Using an efficient and scalable circular RNA (circRNA) platform, we constructed four circRNA vaccines that could induce robust neutralizing antibodies as well as T cell responses by expressing different surface proteins of mpox virus (MPXV), resulting in potent protection against vaccinia virus (VACV) in mice. Strikingly, the combination of the four circular RNA vaccines demonstrated the best protection against VACV challenge among all the tested vaccines. Our study provides a favorable approach for developing MPXV-specific vaccines by using a circular mRNA platform and opens up novel avenues for future vaccine research.

Trnp1 organizes diverse nuclear membrane-less compartments in neural stem cells.

TMF1-regulated nuclear protein 1 (Trnp1) has been shown to exert potent roles in neural development affecting neural stem cell self-renewal and brain folding, but its molecular function in the nucleus is still unknown. Here, we show that Trnp1 is a low complexity protein with the capacity to phase separate. Trnp1 interacts with factors located in several nuclear membrane-less organelles, the nucleolus, nuclear speckles, and condensed chromatin. Importantly, Trnp1 co-regulates the architecture and function of these nuclear compartments in vitro and in the developing brain in vivo. Deletion of a highly conserved region in the N-terminal intrinsic disordered region abolishes the capacity of Trnp1 to regulate nucleoli and heterochromatin size, proliferation, and M-phase length; decreases the capacity to phase separate; and abrogates most of Trnp1 protein interactions. Thus, we identified Trnp1 as a novel regulator of several nuclear membrane-less compartments, a function important to maintain cells in a self-renewing proliferative state.

Synthetic Cells from Droplet-Based Microfluidics for Biosensing and Biomedical Applications.

Synthetic cells function as biological mimics of natural cells by mimicking salient features of cells such as metabolism, response to stimuli, gene expression, direct metabolism, and high stability. Droplet-based microfluidic technology presents the opportunity for encapsulating biological functional components in uni-lamellar liposome or polymer droplets. Verified by its success in the fabrication of synthetic cells, microfluidic technology is widely replacing conventional labor-intensive, expensive, and sophisticated techniques justified by its ability to miniaturize and perform batch production operations. In this review, an overview of recent research on the preparation of synthetic cells through droplet-based microfluidics is provided. Different synthetic cells including lipid vesicles (liposome), polymer vesicles (polymersome), coacervate microdroplets, and colloidosomes, are systematically discussed. Efforts are then made to discuss the design of a variety of microfluidic chips for synthetic cell preparation since the combination of microfluidics with bottom-up synthetic biology allows for reproductive and tunable construction of batches of synthetic cell models from simple structures to higher hierarchical structures. The recent advances aimed at exploiting them in biosensors and other biomedical applications are then discussed. Finally, some perspectives on the challenges and future developments of synthetic cell research with microfluidics for biomimetic science and biomedical applications are provided.

EXO1/P53/SREBP1 axis-regulated lipid metabolism promotes prostate cancer progression.

Prostate cancer (PCa) is one of the most common malignant tumors affecting the male genitourinary system. However, there is currently a lack of effective treatments for patients with advanced prostate cancer, which significantly impacts men's overall health. Exonuclease 1 (EXO1), a protein with mismatch repair and recombination functions, has been found to play a vital role in various diseases. In our study, we discovered that EXO1 acts as a novel biomarker of PCa, which promotes prostate cancer progression by regulating lipid metabolism reprogramming in prostate cancer cells. Mechanistically, EXO1 promotes the expression of SREBP1 by inhibiting the P53 signaling pathway. In summary, our findings suggest that EXO1 regulated intracellular lipid reprogramming through the P53/SREBP1 axis, thus promoting PCa progression. The result could potentially lead to new insights and therapeutic targets for diagnosing and treating PCa.

Requirements on double-cladding Yb-doped fiber for power scaling of diffraction-limited fiber amplifiers.

Requirements on the double-cladding Yb-doped fiber (DCYF) for power scaling of diffraction-limited fiber amplifiers are studied in this paper. By considering the limitations of thermal lens and transverse mode instability separately, it is found that the power scaling limit can be enlarged to more-than 100 kW and 80 kW, respectively, with the proper selection of pump and signal wavelengths. It is also found that the requirements on DCYF strongly depend on the wavelength and brightness of pump light. It is predicted that smaller-than 20-µm core diameter is required to achieve the 20-kW output power, as long as the 976-nm pump brightness can be high enough. The requirements on the inner-cladding diameter and cladding-to-core ratio of DCYF are also investigated.

2 W, 1.5†µm single-mode fiber methane Raman laser pumped by a Yb-doped fiber amplifier.

We report here, to the best of our knowledge, the first 1.5 µm methane-filled fiber Raman laser pumped by a fiber laser. Based on the narrow-linewidth pulsed Yb-doped fiber laser pump source and a 15 m hollow-core fiber filled with 2.5 bar methane, the maximum power of 2.06 W Stokes wave at 1543 nm is obtained. The output laser has a narrow linewidth of 2.3 GHz, and the pulse repetition frequency can be adjusted flexibly. The output shows excellent near-diffraction-limited beam quality with a M2 factor of ∼1.09. This work proves the advantage of the fiber laser pump source with modest peak power and flexible temporal characteristics in 1.5 µm fiber gas Raman laser emission, providing good guidance for generating pulsed fiber source with narrow linewidth and high beam quality.

Femtosecond laser direct writing large-area fiber Bragg grating based on diaphragm shaping.

We propose and demonstrate a new method of direct writing large-area fiber Bragg grating by femtosecond laser through the coating. By adding an adjustable diaphragm before the focusing objective, we can precisely control the length of the refractive index modulation line along the femtosecond laser incident direction up to 29.1 µm. In combination with femtosecond laser scanning fabrication technology, a uniform refractive index modulation plane can be inscribed in the fiber in a single scanning. Based on the plane-by-plane inscription method, we have fabricated a high-quality high-reflectivity fiber Bragg grating and a chirped fiber Bragg grating on 20/400 double-clad fiber core. The reflectivity of both gratings is greater than 99%, and the insertion loss is as low as 0.165 dB and 0.162 dB, respectively. The thermal slope of chirped fiber Bragg grating without any refrigeration is 0.088 °C/W and there is no obvious temperature increase when using the water cooling. Therefore, the fabrication method of large-area fiber Bragg grating based on diaphragm shaping can efficiently fabricate high-quality fiber Bragg grating in the large core diameter fiber, which has an important application prospect in high-power all-fiber oscillators, especially all-fiber oscillators in special wavebands.

Demonstration of hundred-watt-level near-diffraction-limited monolithic fiber laser near 980 nm with step-index double-cladding Yb-doped fiber.

In this paper, a hundred-watt-level near-diffraction-limited step-index Yb-doped fiber (YDF) laser near 980 nm is demonstrated firstly, to the best of our knowledge. By using the 11.7-W 979-nm single-mode seed light, the in-band amplified spontaneous emission (ASE) is well suppressed and the maximum output power of 101.5 W with the beam quality (M2 factor) of 1.285 was obtained. This work does not only propose an effective method for the suppression of in-band ASE, but also provides a cost-effective solution of hundred-Watt-level near-diffraction-limited fiber lasers near 980 nm.

5†kW power-level 1050†nm narrow-linewidth fiber amplifier enabled by biconical-tapered active fiber.

Effective wavelength extension is vital in the applications of high-power narrow-linewidth fiber lasers. In this work, we demonstrate a 5-kW power-level narrow-linewidth fiber amplifier at 1050 nm utilizing a homemade biconical-tapered Yb-doped fiber (BT-YDF). Up to ∼4.96 kW fiber laser is achieved with a 3 dB linewidth of ∼0.54 nm and a beam quality factor of Mx 2 = 1.46, My 2 = 1.6. The experimental comparisons reveal that BT-YDF has the advantages of improving a stimulated Raman scattering threshold and balancing transverse mode instability suppression in the fiber amplifier. This work could provide a good reference for extending the operating wavelength of high-power fiber amplifiers.

Programmable RNA base editing with a single gRNA-free enzyme.

Programmable RNA editing enables rewriting gene expression without changing genome sequences. Current tools for specific RNA editing dependent on the assembly of guide RNA into an RNA/protein complex, causing delivery barrier and low editing efficiency. We report a new gRNA-free system, RNA editing with individual RNA-binding enzyme (REWIRE), to perform precise base editing with a single engineered protein. This artificial enzyme contains a human-originated programmable PUF domain to specifically recognize RNAs and different deaminase domains to achieve efficient A-to-I or C-to-U editing, which achieved 60-80% editing rate in human cells, with a few non-specific editing sites in the targeted region and a low level off-target effect globally. The RNA-binding domain in REWIREs was further optimized to improve editing efficiency and minimize off-target effects. We applied the REWIREs to correct disease-associated mutations and achieve both types of base editing in mice. As a single-component system originated from human proteins, REWIRE presents a precise and efficient RNA editing platform with broad applicability.

Osmotic-Induced Reconfiguration and Activation in Membranized Coacervate-Based Protocells.

The design and construction of synthetic protocells capable of stimuli response and homeostatic regulation is an important challenge for synthetic protobiology. Here, we develop a step toward the construction of model protocells capable of a hypotonic stress-induced volume response that facilitates an increase in membrane permeability and the triggering of endogenous enzyme reactions. We describe a facile self-transformation process for constructing single- or multichambered molecularly crowded protocells based on the osmotic reconfiguration of lipid-coated coacervate droplets into multicompartmentalized coacervate vesicles. Hypotonic swelling broadens membrane permeability and increases transmembrane transport such that protease-based hydrolysis and enzyme cascades can be triggered and enhanced within the protocells by osmotically induced expansion. Specifically, we demonstrate how the enhanced production of nitric oxide (NO) within the swollen coacervate vesicles can be used to induce in vitro blood vessel vasodilation in thoracic artery rings. Our approach provides opportunities for designing reconfigurable model protocells capable of homeostatic volume regulation, dynamic structural reorganization, and adaptive functionality in response to changes in environment osmolarity, and could find applications in biomedicine, cellular diagnostics, and bioengineering.

Selection principle of seed power in high-power narrow linewidth fiber amplifier seeded by a FBGs-based fiber oscillator.

Here, we have experimentally demonstrated the selection principle of the seed power in a narrow linewidth fiber amplifier seeded by fiber oscillator based on a pair of fiber Bragg gratings. During the study on the selection of seed power, the spectral instability of the amplifier is found when a low power seed with bad temporal characteristics is amplified. This phenomenon is thoroughly analyzed from seed itself and the influence of the amplifier. Increasing the seed power or isolating the backward light of amplifier could effectively eliminate the spectral instability. Based on this point, we optimize the seed power and utilize a band pass filter circulator to isolate the backward light and filter the Raman noise. Finally, a 4.2 kW narrow linewidth output power is achieved with signal to noise ratio of 35 dB, which has exceeded the value under the highest output power reported in this type of narrow linewidth fiber amplifiers. This work provides a solution for high power and high signal to noise ratio narrow-linewidth fiber amplifiers seeded by FBGs-based fiber oscillator.

Stimulated Raman scattering of H2 in hollow-core photonics crystal fibers pumped by high-power narrow-linewidth fiber oscillators.

The stimulated Raman scattering (SRS) process in gas-filled hollow-core fiber is mostly used to realize the wavelength conversion, which has the potential to produce narrow-linewidth and high-power fiber laser output. However, limited by the coupling technology, the current research is still at a few watts power level. Here, through the fusion splicing between the end-cap and the hollow-core photonics crystal fiber, several hundred watts pump power can be coupled into the hollow core. Homemade narrow-linewidth continuous wave (CW) fiber oscillators with different 3 dB linewidths are used as the pump sources, then the influences of the pump linewidth and the hollow-core fiber length are studied experimentally and theoretically. As the hollow-core fiber length is 5 m the H2 pressure is 30 bar, 109 W 1st Raman power is obtained with a Raman conversion efficiency 48.5%. This study is significant for the development of high-power gas SRS in hollow-core fibers.

Numerical simulation and observed rotational relaxation in CW and pulsed HBr-filled hollow-core fiber lasers.

Gas-filled hollow-core fiber (HCF) lasers have emerged as a promising technology for generating mid-infrared lasers. A four-energy level system laser model is presented to predict the performance of optically pumped HBr-filled HCF lasers under continuous wave (CW) and pulsed excitations. The steady state condition is considered in CW pumping and the characteristics of simulated population density and power distribution along HCF are investigated. The finite-difference time-domain method is employed in pulsed pumping and the simulated evolutions of pump pulse and laser pulse at different positions along the HCF are studied. In addition, the phenomena of rotational relaxation in HBr-filled HCF lasers are investigated experimentally for the first time, to the best of our knowledge, showing that using the absorption lines away from the strongest absorption lines and tuning the pump wavelength deviating from the center of the absorption line makes the rotational relaxation occur easily. The demonstration is conductive to reveal the underlying mechanism of such gas-filled HCF lasers.

High power mid-infrared fiber amplifier at 3.1†µm by acetylene-filled hollow-core fibers.

We characterized high-power continuous-wave (CW) and pulsed mid-infrared (mid-IR) fiber amplifiers at a wavelength of 3.1 µm in acetylene-filled hollow-core fibers (HCFs) with a homemade seed laser. A maximum CW power of 7.9 W was achieved in a 4.2-m HCF filled with 4-mbar acetylene, which was 11% higher than the power without the seed. The maximum average power of the pulsed laser was 8.6 W (pulse energy of 0.86 µJ) at 7-mbar acetylene pressure, a 16% increase over the power without the seed. To the best of our knowledge, backward characteristics are reported for the first time for fiber gas lasers, and the backward power accounted for less than 5% of the forward power. The optimum acetylene pressure and HCF length for the highest mid-IR output are discussed based on theoretical simulations. This study provides significant guidance for high-power mid-infrared (mid-IR) output in gas-filled HCFs.

Raman suppression in high-power fiber oscillators by femtosecond-written chirped and tilted fiber Bragg gratings.

Realizing stimulated Raman scattering (SRS) suppression is a key topic for high-power fiber lasers. Here, we report an effective and simple strategy for SRS suppression using chirped and tilted fiber Bragg gratings (CTFBGs) in high-power fiber oscillators while maintaining the compactness and stability of the system. The CTFBG is inserted on the side of a cavity mirror FBG without cutting the gain fiber. To improve power handling capability, the CTFBG and cavity mirror FBGs are inscribed by femtosecond (fs) lasers. The optimal SRS suppression effect can be realized when the CTFBG is inserted into the resonant cavity and on the side of the output coupler FBG. The SRS threshold is increased by approximately 11% with an SRS suppression ratio of nearly 14 dB. Moreover, the output power of the fiber oscillator is improved to 3.5 kW, which is the maximum power achieved in fiber oscillators with SRS suppression using CTFBGs, to the best of our knowledge. The temperature of the air-cooled CTFBG is 50.2 °C, which has the potential to handle higher power. This work provides new insights for suppressing SRS in fiber oscillators, promoting the application of CTFBGs in high-power lasers.

2.3-µm single-frequency Tm: ZBLAN fiber amplifier with output power of 1.41 W.

We present here the first watt-level single-frequency thulium-doped ZBLAN fiber amplifier system operating at a wavelength of 2.3 µm. Continuous-wave output of up to 1.41 W was generated from a two-stage Tm: ZBLAN fiber amplifier with direct ground-state pumping at 793 nm. Seeded by a single-frequency distributed feedback diode laser at 2332 nm, the thulium-doped ZBLAN fiber amplifier emitted a laser with linewidth no more than 10 MHz at maximal output power. This study examines the impact of a 2.3-µm seed on the competitive laser transition of 2 µm. The findings indicate that direct pumping of a Tm fiber amplifier holds the potential for achieving higher power output within the 2.3-µm band.