Upconversion-pumped femtosecond thulium laser at 2309†nm mode-locked by a GaSb-based SESAM.

We report on a femtosecond thulium laser operating on the 3H4 → 3H5 transition with upconversion pumping around 1 µm and passively mode-locked by a GaSb-based SEmiconductor Saturable Absorber Mirror (SESAM). This laser employs a 6 at.% Tm:LiYF4 laser crystal and a polarization maintaining Yb-fiber master oscillator power amplifier at 1043 nm as a pump source addressing the 3F4 → 3F2,3 excited-state absorption transition of Tm3+ ions. In the continuous-wave regime, the Tm-laser generates 616 mW at ∼2313 nm with a slope efficiency of 10.0% (vs. the incident pump power) and a linear polarization (π). By implementing a type-I SESAM with a single ternary strained In0.33Ga0.67Sb quantum well embedded in GaSb for sustaining and stabilizing the soliton pulse shaping, the self-starting mode-locked Tm-laser generated pulses as short as 870 fs at a central wavelength of 2309.4 nm corresponding to an average output power of 208 mW at a pulse repetition rate of 105.08 MHz and excellent mode-locking stability. The output power was scaled to 450 mW at the expense of a longer pulse duration of 1.93 ps. The nonlinear parameters of the SESAM are also reported.

Inhibition of VHL by VH298 Accelerates Pexophagy by Activation of HIF-1α in HeLa Cells.

Autophagy is a pivotal biological process responsible for maintaining the homeostasis of intracellular organelles. Yet the molecular intricacies of peroxisomal autophagy (pexophagy) remain largely elusive. From a ubiquitin-related chemical library for screening, we identified several inhibitors of the Von Hippel-Lindau (VHL) E3 ligase, including VH298, thereby serving as potent inducers of pexophagy. In this study, we observed that VH298 stimulates peroxisomal degradation by ATG5 dependently and escalates the ubiquitination of the peroxisomal membrane protein ABCD3. Interestingly, the ablation of NBR1 is similar to the curtailed peroxisomal degradation in VH298-treated cells. We also found that the pexophagy induced by VH298 is impeded upon the suppression of gene expression by the translation inhibitor cycloheximide. Beyond VHL inhibition, we discovered that roxadustat, a direct inhibitor of HIF-α prolyl hydroxylase, is also a potent inducer of pexophagy. Furthermore, we found that VH298-mediated pexophagy is blocked by silencing HIF-1α. In conclusion, our findings suggest that VH298 promotes pexophagy by modulating VHL-mediated HIF-α transcriptional activity.

Reliability of the In Silico Prediction Approach to In Vitro Evaluation of Bacterial Toxicity.

Several pathogens that spread through the air are highly contagious, and related infectious diseases are more easily transmitted through airborne transmission under indoor conditions, as observed during the COVID-19 pandemic. Indoor air contaminated by microorganisms, including viruses, bacteria, and fungi, or by derived pathogenic substances, can endanger human health. Thus, identifying and analyzing the potential pathogens residing in the air are crucial to preventing disease and maintaining indoor air quality. Here, we applied deep learning technology to analyze and predict the toxicity of bacteria in indoor air. We trained the ProtBert model on toxic bacterial and virulence factor proteins and applied them to predict the potential toxicity of some bacterial species by analyzing their protein sequences. The results reflect the results of the in vitro analysis of their toxicity in human cells. The in silico-based simulation and the obtained results demonstrated that it is plausible to find possible toxic sequences in unknown protein sequences.

Depletion of HNRNPA1 induces peroxisomal autophagy by regulating PEX1 expression.

Peroxisomes play an essential role in cellular homeostasis by regulating lipid metabolism and the conversion of reactive oxygen species (ROS). Several peroxisomal proteins, known as peroxins (PEXs), control peroxisome biogenesis and degradation. Various mutations in the PEX genes are genetic causes for the development of inheritable peroxisomal-biogenesis disorders, such as Zellweger syndrome. Among the peroxins, PEX1 defects are the most common mutations in Zellweger syndrome. PEX1 is an AAA-ATPase that regulates the recycling of PEX5, which is essential for importing peroxisome matrix proteins. However, the post-transcriptional regulation of PEX1 is largely unknown. Here, we showed that heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1) controls PEX1 expression. In addition, we found that depletion of HNRNPA1 induces autophagic degradation of peroxisome, which is blocked in ATG5-knockout cells. In addition, depletion of HNRNPA1 increased peroxisomal ROS levels. Inhibition of the generation of peroxisomal ROS by treatment with NAC significantly suppressed pexophagy in HNRNPA1-deficient cells. Taken together, our results suggest that depletion of HNRNPA1 increases peroxisomal ROS and pexophagy by downregulating PEX1 expression.

Sub-100 fs mode-locked Tm:CLTGG laser.

We report on the first sub-100 fs mode-locked laser operation of a Tm3+-doped disordered calcium lithium tantalum gallium garnet (Tm:CLTGG) crystal. Soliton mode-locking was initiated and stabilized by a transmission-type single-walled carbon nanotube saturable absorber. Pulses as short as 69 fs were achieved at a central wavelength of 2010.4 nm with an average power of 28 mW at a pulse repetition rate of ∼87.7 MHz. In the sub-100 fs regime, the maximum average output power amounted to 103 mW.

SWCNT-SA mode-locked Tm,Ho:LCLNGG laser.

Sub-100 fs pulse generation from a passively mode-locked Tm,Ho-codoped cubic multicomponent disordered garnet laser at ∼2 µm is demonstrated. A single-walled carbon nanotube saturable absorber is implemented to initiate and stabilize the soliton mode-locking. The Tm,Ho:LCLNGG (lanthanum calcium lithium niobium gallium garnet) laser generated pulses as short as 63 fs at a central wavelength of 2072.7 nm with an average output power of 63 mW at a pulse repetition rate of ∼102.5 MHz. Higher average output power of 121 mW was obtained at the expense of longer pulse duration (96 fs) at 2067.6 nm using higher output coupling. To the best of our knowledge, this is the first report on mode-locked operation of the Tm,Ho:LCLNGG crystal.

Can Designer Indels Be Tailored by Gene Editing?: Can Indels Be Customized?

Genome editing with engineered nucleases (GEENs) introduce site-specific DNA double-strand breaks (DSBs) and repairs DSBs via nonhomologous end-joining (NHEJ) pathways that eventually create indels (insertions/deletions) in a genome. Whether the features of indels resulting from gene editing could be customized is asked. A review of the literature reveals how gene editing technologies via NHEJ pathways impact gene editing. The survey consolidates a body of literature that suggests that the type (insertion, deletion, and complex) and the approximate length of indel edits can be somewhat customized with different GEENs and by manipulating the expression of key NHEJ genes. Structural data suggest that binding of GEENs to DNA may interfere with binding of key components of DNA repair complexes, favoring either classical- or alternative-NHEJ. The hypotheses have some limitations, but if validated, will enable scientists to better control indel makeup, holding promise for basic science and clinical applications of gene editing. Also see the video abstract here https://youtu.be/vTkJtUsLi3w.

CRISPR/Cas9 cleavages in budding yeast reveal templated insertions and strand-specific insertion/deletion profiles.

Harnessing CRISPR-Cas9 technology provides an unprecedented ability to modify genomic loci via DNA double-strand break (DSB) induction and repair. We analyzed nonhomologous end-joining (NHEJ) repair induced by Cas9 in budding yeast and found that the orientation of binding of Cas9 and its guide RNA (gRNA) profoundly influences the pattern of insertion/deletions (indels) at the site of cleavage. A common indel created by Cas9 is a 1-bp (+1) insertion that appears to result from Cas9 creating a 1-nt 5' overhang that is filled in by a DNA polymerase and ligated. The origin of +1 insertions was investigated by using two gRNAs with PAM sequences located on opposite DNA strands but designed to cleave the same sequence. These templated +1 insertions are dependent on the X-family DNA polymerase, Pol4. Deleting Pol4 also eliminated +2 and +3 insertions, which are biased toward homonucleotide insertions. Using inverted PAM sequences, we also found significant differences in overall NHEJ efficiency and repair profiles, suggesting that the binding of the Cas9:gRNA complex influences subsequent NHEJ processing. As with events induced by the site-specific HO endonuclease, CRISPR-Cas9-mediated NHEJ repair depends on the Ku heterodimer and DNA ligase 4. Cas9 events are highly dependent on the Mre11-Rad50-Xrs2 complex, independent of Mre11's nuclease activity. Inspection of the outcomes of a large number of Cas9 cleavage events in mammalian cells reveals a similar templated origin of +1 insertions in human cells, but also a significant frequency of similarly templated +2 insertions.

Primary Ciliogenesis by 2-Isopropylmalic Acid Prevents PM2.5-Induced Inflammatory Response and MMP-1 Activation in Human Dermal Fibroblasts and a 3-D-Skin Model.

Particulate matters (PMs) increase oxidative stress and inflammatory response in different tissues. PMs disrupt the formation of primary cilia in various skin cells, including keratinocytes and melanocytes. In this study, we found that 2-isopropylmalic acid (2-IPMA) promoted primary ciliogenesis and restored the PM2.5-induced dysgenesis of primary cilia in dermal fibroblasts. Moreover, 2-IPMA inhibited the generation of excessive reactive oxygen species and the activation of stress kinase in PM2.5-treated dermal fibroblasts. Further, 2-IPMA inhibited the production of pro-inflammatory cytokines, including IL-6 and TNF-α, which were upregulated by PM2.5. However, the inhibition of primary ciliogenesis by IFT88 depletion reversed the downregulated cytokines by 2-IPMA. Moreover, we found that PM2.5 treatment increased the MMP-1 expression in dermal fibroblasts and a human 3-D-skin model. The reduced MMP-1 expression by 2-IPMA was further reversed by IFT88 depletion in PM2.5-treated dermal fibroblasts. These findings suggest that 2-IPMA ameliorates PM2.5-induced inflammation by promoting primary ciliogenesis in dermal fibroblasts.

2-IPMA Ameliorates PM2.5-Induced Inflammation by Promoting Primary Ciliogenesis in RPE Cells.

Primary cilia mediate the interactions between cells and external stresses. Thus, dysregulation of primary cilia is implicated in various ciliopathies, e.g., degeneration of the retina caused by dysregulation of the photoreceptor primary cilium. Particulate matter (PM) can cause epithelium injury and endothelial dysfunction by increasing oxidative stress and inflammatory responses. Previously, we showed that PM disrupts the formation of primary cilia in retinal pigment epithelium (RPE) cells. In the present study, we identified 2-isopropylmalic acid (2-IPMA) as a novel inducer of primary ciliogenesis from a metabolite library screening. Both ciliated cells and primary cilium length were increased in 2-IPMA-treated RPE cells. Notably, 2-IPMA strongly promoted primary ciliogenesis and restored PM2.5-induced dysgenesis of primary cilia in RPE cells. Both excessive reactive oxygen species (ROS) generation and activation of a stress kinase, JNK, by PM2.5 were reduced by 2-IPMA. Moreover, 2-IPMA inhibited proinflammatory cytokine production, i.e., IL-6 and TNF-α, induced by PM2.5 in RPE cells. Taken together, our data suggest that 2-IPMA ameliorates PM2.5-induced inflammation by promoting primary ciliogenesis in RPE cells.

The Benefits of Adjuvant Trastuzumab for HER-2-Positive Salivary Gland Cancers.

BACKGROUND: Although high-grade salivary gland cancers (SGCs) often express androgen receptor (AR) and/or HER-2/neu, therapeutically targeting these receptors in SGC remains investigational. We investigated the prevalence of receptor expression and the benefit of adjuvant HER-2 directed therapy in the high-risk postoperative setting and explored the clinical utility of sequentially targeting these receptors in the setting of advanced disease. MATERIALS AND METHODS: We clinically annotated 95 patients with SGC (excluding adenoid cystic carcinoma) treated at our institution from 2002 to 2019 and recorded AR, HER-2/neu status, and tumor genomic profiling results when available. Clinicopathologic information was then integrated with outcomes. RESULTS: Of 95 patients, most had high-risk histologies, with salivary duct carcinoma (SDC) as the most frequent diagnosis (43, 45%). Thirty-five (37%) experienced recurrence (51% SDC). HER-2/neu was positive (1-3+) by immunostaining in 34 of 52 (65%) evaluable cases. There was no difference in survival based on HER-2/neu or AR expression. Nine of 17 (53%) patients with HER-2+ SDC received adjuvant chemoradiation with trastuzumab. Median disease-free survival (DFS) and overall survival (OS) were longer among patients with HER-2/neu 3+ staining tumors who received adjuvant trastuzumab versus those who did not (DFS, 117 vs. 9 months; p = .02; OS, 74 vs. 43 months; p = .02), with no difference among other HER-2/neu subgroups (0-2+). Two of nine (22%) patients treated with adjuvant trastuzumab demonstrated recurrence, both with low HER-2/neu staining intensity (1+). Longer time to recurrence (hazard ratio, 0.94; p = .01) predicted improved outcomes. Both androgen deprivation and HER-2-directed therapies had clinical benefit beyond the first-line metastatic setting, with partial response observed beyond second-line use. CONCLUSION: Although prospective data are lacking, the use of adjuvant trastuzumab in high-risk patients with SGC appears beneficial, particularly among patients with tumors exhibiting HER-2/neu 3+ immunostaining. IMPLICATIONS FOR PRACTICE: Results of this study showed an improved disease-free and overall survival in patients treated with adjuvant trastuzumab for high-risk salivary gland cancers with strong HER-2/neu staining intensity. Following recurrence or metastatic spread, sequential HER-2, and androgen-directed therapies may benefit certain patients with salivary gland cancer.

Ursolic acid inhibits pigmentation by increasing melanosomal autophagy in B16F1 cells.

Melanosomes are specialized membrane-bound organelles that are involved in melanin synthesis. Unlike melanosome biogenesis, the melanosome degradation pathway is poorly understood. Among the cellular processes, autophagy controls degradation of intracellular components by cooperating with lysosomes. In this study, we showed that ursolic acid inhibits skin pigmentation by promoting melanosomal autophagy, or melanophagy, in melanocytes. We found that B16F1 cells treated with ursolic acid suppressed alpha-melanocyte stimulating hormone (α-MSH) stimulated increase in melanin content and activated autophagy. In addition, we found that treatment with ursolic acid promotes melanosomal degradation, and bafilomycin A1 inhibition of autophagosome-lysosome fusion blocked the removal of melanosomes in α-MSH-stimulated B16F1 cells. Furthermore, depletion of the autophagy-related gene 5 (ATG5) resulted in significant suppression of ursolic acid-mediated anti-pigmentation activity and autophagy in α-MSH-treated B16F1 cells. Taken together, our results suggest that ursolic acid inhibits skin pigmentation by increasing melanosomal degradation in melanocytes.

Transition of pulsed operation from Q-switching to continuous-wave mode-locking in a Yb:KLuW waveguide laser.

We report on the diverse pulsed operation regimes of a femtosecond-laser-written Yb:KLuW channel waveguide laser emitting near 1040 nm. By the precise position tuning of a carbon-nanotube-coated saturable absorber (SA) mirror, the transition of the pulsed operation from Q-switching, Q-switched mode-locking and finally sub-GHz continuous-wave mode-locking are obtained based on the interplay of dispersion and mode area control. The Q-switched pulses exhibit typical fast SA Q-switched pulse characteristics depending on absorbed pump powers. In the Q-switched mode-locking, amplitude modulations of the mode-locked pulses on the Q-switched envelope are observed. The radio-frequency spectrum represents the coexistence of Q-switching and mode-locking signals. In the purely mode-locked operation, the waveguide laser generates 2.05-ps pulses at 0.5 GHz.

Spectroscopy and diode-pumped laser operation of transparent Tm:Lu3Al5O12 ceramics produced by solid-state sintering.

A transparent Tm:Lu3Al5O12 ceramic is fabricated by solid-state reactive sintering at 1830 °C for 30 h using commercial α-Al2O3 and Lu2O3/Tm2O3 powders and sintering aids - MgO and TEOS. The ceramic belongs to the cubic system and exhibits a close-packed structure (mean grain size: 21 µm). The in-line transmission at ∼1 µm is 82.6%, close to the theoretical limit. The spectroscopic properties of the ceramic are studied in detail. The maximum stimulated-emission cross-section is 2.37×10-21 cm2 at 1749nm and the radiative lifetime of the 3F4 state is about 10 ms. The modified Judd-Ofelt theory accounting for configuration interaction is applied to determine the transition probabilities of Tm3+, yielding the intensity parameters Ω2 = 2.507, Ω4 = 1.236, Ω6 = 1.340 [10-20 cm2] and α = 0.196×10-4 cm. The effect of excited configurations on lower-lying interconnected states with the same J quantum number is discussed. First laser operation is achieved under diode-pumping at 792 nm. A 4 at.% Tm:Lu3Al5O12 ceramic laser generated 3.12 W at 2022-2035nm with a slope efficiency of 60.2%. The ceramic is promising for multi-watt lasers at >2 µm.

Single-walled carbon-nanotube saturable absorber assisted Kerr-lens mode-locked Tm:MgWO4 laser.

We demonstrate sub-100-fs Kerr-lens mode-locking of a Tm:MgWO4 laser emitting at ∼2µm assisted by a single-walled carbon-nanotube saturable absorber. A maximum average output power of 100 mW is achieved with pulse duration of 89 fs at a pulse repetition rate of ∼86MHz. The shortest pulse duration derived from frequency-resolved optical gating amounts to 76 fs at 2037 nm, corresponding to nearly bandwidth-limited pulses. To the best of our knowledge, these are the shortest pulses generated from any Tm-doped tungstate crystal and the first report on saturable absorber assisted Kerr-lens mode-locking of a Tm laser at ∼2µm.

Loss of RNA binding protein, human antigen R enhances mitochondrial elongation by regulating Drp1 expression in SH-SY5Y cells.

Mitochondria are essential for providing the energy necessary for neuronal function. Dysregulation of mitochondrial dynamics has been linked with the pathogenesis of many neurodegenerative diseases. Dynamin related protein 1 (Drp1) participates in fission activity in the mitochondria, and post-translational modifications to Drp1 modulate complex mitochondrial dynamics. However, the regulation of Drp1 at the post-transcriptional level remains poorly understood. In this study, we found that the RNA-binding protein Hu antigen R (HuR) post-transcriptionally regulates Drp1 expression. HuR interacts with Drp1 mRNA at its 3' untranslated region. Depletion of HuR reduces Drp1 expression, which leads to mitochondrial elongation in SH-SY5Y neuroblastoma cells. In contrast, ectopic expression of HuR enhances Drp1 expression, which promotes mitochondrial fragmentation in response to treatment with the mitochondrial complex 1 inhibitor MPP+. In addition, depletion of HuR suppressed the generation of mitochondrial ROS and cytotoxicity in MPP+ treated cells. Taken together, these findings suggest that HuR controls mitochondrial morphology via regulation of Drp1.

67-fs pulse generation from a mode-locked Tm,Ho:CLNGG laser at 2083 nm.

We report on a mode-locked Tm,Ho:CLNGG laser emitting in the 2 µm spectral range using single-walled carbon nanotubes (SWCNTs) as a saturable absorber (SA). Pulses with duration of 98 fs are generated at 99.28 MHz repetition rate with an average output power of 123 mW, yielding a pulse energy of 1.24 nJ. Using a 0.5% output coupling, pulses as short as 67 fs, i.e., 10 optical cycles, are produced after extracavity compression with a 3-mm-thick ZnS plate.

Comparative study of Yb:KYW planar waveguide lasers Q-switched by direct- and evanescent-field interaction with carbon nanotubes.

Both direct- and evanescent-field interactions with carbon nanotubes (CNTs) are applied to achieve stable Q-switched operation of Yb:KYW planar waveguide lasers. The performance characteristics were investigated in a same cavity configuration and analyzed in detail in the following three cases, CNTs deposited onto end mirror (M-coating), output coupler (OC-coating) and top surface of the planar waveguide (WG-coating). Maximum output powers, repetition rates, and minimum pulse durations are 61 mW, 1103 kHz and 215 ns for OC-coating, 39 mW, 1052 kHz and 275 ns for WG-coating, and 26 mW, 1119 kHz and 217 ns for M-coating, respectively. From the calculation of the configuration-dependent stability range, the beam size and the electric field distribution in the Yb:KYW planar waveguide, it is confirmed that the evanescent-field interaction scheme makes stable Q-switching possible with much lower intensities at saturable absorber compared to the direct-field interaction scheme in the presented waveguide laser operation.

21 fs Cr:LiSAF laser mode locked with a single-walled carbon nanotube saturable absorber.

We report the shortest femtosecond pulses directly generated from a solid-state laser that is mode locked by using a single-walled carbon nanotube saturable absorber (SWCNT-SA). In the experiments, we used a 660 nm diode-pumped, low-threshold extended-cavity Cr:LiSAF laser operating around 850 nm with a repetition rate of 47.9 MHz. The SWCNT-SA mode-locked Cr:LiSAF laser produced 21 fs pulses with a time-bandwidth product of 0.56 by using only 210 mW of pump power. Pump-probe spectroscopy measurements showed that the SWCNT-SA exhibited saturable absorption with slow and fast decay times of 2.7 ps and 0.4 ps. The single-pass modulation depth and saturation fluence of the SWCNT-SA were further determined as 0.3% and 45 µJ/cm2 at the pump wavelength of 850 nm.

Structural analysis of substrate recognition by glucose isomerase in Mn2+ binding mode at M2 site in S. rubiginosus.

Glucose isomerase (GI) catalyzes the reversible enzymatic isomerization of d-glucose and d-xylose to d-fructose and d-xylulose, respectively. This is one of the most important enzymes in the production of high-fructose corn syrup (HFCS) and biofuel. We recently determined the crystal structure of GI from S. rubiginosus (SruGI) complexed with a xylitol inhibitor in one metal binding mode. Although we assessed inhibitor binding at the M1 site, the metal binding at the M2 site and the substrate recognition mechanism for SruGI remains the unclear. Here, we report the crystal structure of the two metal binding modes of SruGI and its complex with glucose. This study provides a snapshot of metal binding at the SruGI M2 site in the presence of Mn2+, but not in the presence of Mg2+. Metal binding at the M2 site elicits a configuration change at the M1 site. Glucose molecule can only bind to the M1 site in presence of Mn2+ at the M2 site. Glucose and Mn2+ at the M2 site were bridged by water molecules using a hydrogen bonding network. The metal binding geometry of the M2 site indicates a distorted octahedral coordination with an angle of 55-110°, whereas the M1 site has a relatively stable octahedral coordination with an angle of 85-95°. We suggest a two-step sequential process for SruGI substrate recognition, in Mn2+ binding mode, at the M2 site. Our results provide a better understanding of the molecular role of the M2 site in GI substrate recognition.