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Abstract Recombinant proteins are gaining increasing popularity for treating human diseases. The clinical effectiveness of recombinant proteins is directly related to their biological activity, which is an important indicator in drug development and quality control. However, certain recombinant proteins have unclear or complex signal pathways, making detecting their activity in vitro difficult. For instance, recombinant human endostatin (endostatin), a new antitumor drug developed in China, lacks a sensitive and stable assay for its biological activity since being market approval. To address this issue, we performed a genome‐wide screening of immortalized human umbilical vein endothelial cells (HUVECs) using a CRISPR/Cas9 knockout library containing 20,000 targeted genes. We identified two potential endostatin‐resistant genes, NEPSPP and UTS2, and successfully constructed a highly sensitive cell line, HUVEC‐UTS2‐3#, by knocking down the UTS2 gene. Based on the optimized parameters of HUVEC‐UTS2‐3# cells, we established a new method for detecting the biological activity of endostatin. The method was validated, and it produced results consistent with primary HUVEC cells but with higher sensitivity and more stable data. The use of gene‐editing technology provides a novel solution for detecting the biological activity of recombinant proteins that other methods cannot detect.

Recombinant adeno-associated virus (rAAV) has emerged as a prominent vector for in vivo gene therapy, owing to its distinct advantages. Accurate determination of the rAAV genome titer is crucial for ensuring the safe and effective administration of clinical doses. The evolution of the rAAV genome titer assay from quantitative PCR (qPCR) to digital PCR (dPCR) has enhanced accuracy and precision, yet practical challenges persist. This study systematically investigated the impact of various operational factors on genome titration in a single-factor manner, aiming to address potential sources of variability in the quantitative determination process. Our findings revealed that a pretreatment procedure without genome extraction exhibits superior precision compared with titration with genome extraction. Additionally, notable variations in titration results across different brands of dPCR instruments were documented, with relative standard deviation (RSD) reaching 23.47% for AAV5 and 11.57% for AAV8. Notably, optimal operations about DNase I digestion were identified; we thought treatment time exceeding 30 min was necessary, and there was no need for thermal inactivation after digestion. And we highlighted that thermal capsid disruption before serial dilution substantially affected AAV genome titers, causing a greater than ten-fold decrease. Conversely, this study found that additive components of dilution buffer are not significant contributors to titration variations. Furthermore, we found that repeated freeze–thaw cycles significantly compromised AAV genome titers. In conclusion, a comprehensive dPCR titration protocol, incorporating insights from these impact factors, was proposed and successfully tested across multiple serotypes of AAV. The results demonstrate acceptable variations, with the RSD consistently below 5.00% for all tested AAV samples. This study provides valuable insights to reduce variability and improve the reproducibility of AAV genome titration using dPCR.

The kinematic information of the Radcliffe wave (RW) is essential for determining its existence and gaining insights into its origin and evolution. In this work, we present an accurate measurement of the vertical velocity ( V _Z ) of the RW by incorporating the radial velocity (RV) measures through two methods, which is crucial but was neglected previously. First, the velocities are measured toward young stars, using their RV measurements from APOGEE-2 and proper motion measurements from Gaia DR3. Second, we combine RV measurements toward clouds with proper motion measurements of associated young stellar objects to determine the vertical velocities of the clouds. The results reveal that the oscillations in V _Z are not synchronous with the vertical coordinate Z , which differs from the conclusions of previous studies. Instead, we find a 5 km s ^−1 kpc ^−1 gradient in V _Z along the RW, exhibiting a dipole-like pattern. Consequently, the kinematic arrangement does not show a corresponding coherence with the spatial arrangement, bringing the RW model into question.

C _4 H and c -C _3 H _2 , as unsaturated hydrocarbon molecules, are important for forming large organic molecules in the interstellar medium. We present mapping observations of C _4 H ( N = 9−8) lines, c -C _3 H _2 ( J _Ka _, _Kb = 2 _1,2 –1 _0,1 ), and H ^13 CO ^+ ( J = 1−0) toward 19 nearby cold molecular cores in the Milky Way with the IRAM 30 m telescope. C _4 H 9–8 was detected in 13 sources, while c -C _3 H _2 was detected in 18 sources. The widely existing C _4 H and c -C _3 H _2 molecules in cold cores provide material to form large organic molecules. Different spatial distributions between C _4 H 9–8 and c -C _3 H _2 2–1 were found. The relative abundances of these three molecules were obtained under the assumption of local thermodynamic equilibrium conditions with a fixed excitation temperature. The abundance ratio of C _4 H to c -C _3 H _2 ranged from 0.34 ± 0.09 in G032.93+02 to 4.65 ± 0.50 in G008.67+22. A weak correlation between C _4 H/H ^13 CO ^+ and c -C _3 H _2 /H ^13 CO ^+ abundance ratios was found, with a correlation coefficient of 0.46, which indicates that there is no tight astrochemical connection between C _4 H and c -C _3 H _2 molecules.

Dense outflowing gas, traced by transitions of molecules with large dipole moment, is important for understanding mass loss and feedback of massive star formation. HCN 3-2 and HCO ^+ 3-2 are good tracers of dense outflowing molecular gas, which are closely related to active star formation. In this study, we present on-the-fly mapping observations of HCN 3-2 and HCO ^+ 3-2 toward a sample of 33 massive star-forming regions using the 10-m Submillimeter Telescope. With the spatial distribution of line wings of HCO ^+ 3-2 and HCN 3-2, outflows are detected in 25 sources, resulting in a detection rate of 76%. The optically thin H ^13 CN and H ^13 CO ^+ 3-2 lines are used to identify line wings as outflows and estimate core mass. The mass M _out , momentum P _out , kinetic energy E _K , force F _out , and mass-loss rate ${\dot{M}}_{{\rm{out}}}$ of outflow and core mass, are obtained for each source. A sublinear tight correlation is found between the mass of dense molecular outflow and core mass, with an index of ∼0.8 and a correlation coefficient of 0.88.

Deuterated molecules are valuable probes for investigating the evolution and the kinematics in the earliest stages of star formation. In this study, we conduct a comprehensive investigation by performing a single-point survey of 101 starless clump candidates, and carrying out on-the-fly (OTF) observations of 11 selected sources, focusing on deuterated molecular lines using the IRAM 30 m telescope. In the single-point observation, we make 46 detections for DCO ^+ J = 1−0, 12 for DCN J = 1−0, 51 for DNC J = 1−0, 7 for N _2 D ^+ J = 1−0, 20 for DCO ^+ J = 2−1, and 10 for DCN J = 2−1. The starless clump candidates with deuterated molecule detections exhibit lower median kinetic temperatures and a narrower H _2 CO (1 _(0,1) −0 _(0,0) ) median full width at half maximum compared to those without such detections, while simultaneously displaying similar median values of 1.1 mm intensity, mass, and distance. Furthermore, our OTF observations reveal that deuterated molecules predominantly have peaks near the 1.1 mm continuum peaks, with the DCO ^+ J = 1−0 emission demonstrating higher intensity in the deuterated peak region compared to the DCN and DNC J = 1−0 emissions. Additionally, the majority of emissions from deuterated molecules and ^13 C isotopologues exhibit peak positions close to those of the 1.1 mm continuum peaks. By analyzing the 20″ × 20″ regions with strongest deuterated emissions in the OTF observations, we estimated deuterated abundances of 0.004−0.045, 0.011−0.040, and 0.004−0.038 for D _frac (HCN), D _frac (HCO ^+ ), and D _frac (HNC), respectively. The differential detection of deuterated molecular lines in our OTF observations could be attributed to variations in critical densities and formation pathways.

Peptide-like molecules, which have a close connection with the origin of life, have been detected in the Universe. Mapping observations of HCONH _2 and CH _3 CONH _2 , two of the simplest peptide-like molecules, are performed toward the Sagittarius B2 (Sgr B2) complex with the IRAM 30 m telescope. Seven transitions of HCONH _2 and five transitions of CH _3 CONH _2 are used in the analysis. The spatial distributions of the excitation temperature and column density of HCONH _2 in the molecular envelope of Sgr B2 are obtained by rotation diagrams. Assuming the same excitation temperature of HCONH _2 , the column densities of CH _3 CONH _2 are also calculated. The results show that the excitation temperature ranges from 6 to 46 K in the molecular envelope of Sgr B2. The abundance ratios between HCONH _2 and CH _3 CONH _2 are calculated to explore the relationship between them, as are those between HCONH _2 and HNCO. The abundance ratio of CH _3 CONH _2 /HCONH _2 varies from 10% to 20%, while that of HCONH _2 /HNCO ranges from 1.5% to 10%. CH _3 CONH _2 is enhanced with respect to HCONH _2 in the northwest region of Sgr B2. One transition of H ^13 CONH _2 is detected toward 12 positions of Sgr B2, from which a ^12 C/ ^13 C ratio of 28.7 is obtained. A time-dependent chemical model with a short-duration X-ray burst is used to explain the observed abundances of HCONH _2 and CH _3 CONH _2 , with the best-fitting result at T _dust = 53–56 K. More chemical reactions are required to be included in the model since the modeled abundance is lower than the observed one at the observed T _dust .

Urbanization improves the living environment of billions of people but negatively impacts the local carbon cycle as it encroaches on ecosystems. We analyzed this dual effect of urbanization by quantifying linkages between several indicators -- urbanization-induced terrestrial carbon storage loss (uTCSL), per capita urban area (PCUA), and urban land use efficiency (LCRPGR) -- within the Hohhot-Baotou-Ordos (HBO) region in 1990-2100. Results show that the growth of 4.45 million urban residents and 420.70 km² urban area led to a uTCSL of 815.74 GgC in the HBO region in 1990-2020. The uTCSL contributed to improving the urban crowding indicated by PCUA but was subject to LCRPGR. The PCUA decreased by -33.75 m²/person under high land use efficiency (LCRPGR<1) in 1990-2010. Although the PCUA increased by 23.59 m²/person from 2010 to 2020, it resulted in a 2.08-fold increase in uTCSL compared to 1990-2010, with each additional 1 m² increase in PCUA being associated with carbon storage loss of 24.50 GgC. Our projections indicate that regulating LCRPGR twenty years before the urban population reaches its peak could benefit both uTCSL and PCUA. This study provides valuable insights for urban planning aligned with the UN 2030 Agenda, especially for arid and semi-arid cities. [ABSTRACT FROM AUTHOR]