How do antimicrobial peptides disrupt the lipopolysaccharide membrane leaflet of Gram-negative bacteria?

HYPOTHESIS: It is widely regarded that antimicrobial peptides (AMPs) kill bacteria by physically disrupting microbial membranes and causing cytoplasmic leakage, but it remains unclear how AMPs disrupt the outer membrane (OM) of Gram-negative bacteria (GNB) and then compromise the inner membrane. We hypothesise that different AMPs impose different structural disruptions, with direct implications to their antimicrobial efficacies. EXPERIMENTS: The antimicrobial activities of three typical AMPs, including the designed short AMP, G3, and two natural AMPs, melittin and LL37, against E. coli and their haemolytic activities were studied. Lipopolysaccharide (LPS) and anionic di-palmitoyl phosphatidyl glycerol (DPPG) monolayer models were constructed to mimic the outer membrane and inner membrane leaflets of Gram-negative bacteria. The binding and penetration of AMPs to the model lipid monolayers were systematically studied by neutron reflection via multiple H/D contrast variations. FINDING: G3 has relatively high antimicrobial activity, low cytotoxicity, and high proteolytic stability, whilst melittin has significant haemolysis and LL37 has weaker antimicrobial activity. G3 could rapidly lyse LPS and DPPG monolayers within 10-20 min. In contrast, melittin was highly active against the LPS membrane, but the dynamic process lasted up to 80 min, with excessive stacking in the OM. LL37 caused rather weak destruction to LPS and DPPG monolayers, leading to massive adsorption on the membrane surface without penetrating the lipid tail region. These findings demonstrate that the rationally designed AMP G3 was well optimised to impose most effective destruction to bacterial membranes, consistent with its highest bactericidal activity. These different interfacial structural features associated with AMP binding shed light on the future development of active and biocompatible AMPs for infection and wound treatments.

Acyl chain length tuning improves antimicrobial potency and biocompatibility of short designed lipopeptides.

HYPOTHESIS: Designed antimicrobial lipopeptides (ALPs) offer the attractive benefits of short peptide sequences and flexible tuning of amphiphilicity by altering the acyl chain length. These lipopeptides kill microbes by forming intriguing in-membrane nanostructures and causing the leakage of internal contents. However, how subtle differences in the molecular structures of the lipopeptides affect their antimicrobial efficacy and biocompatibility to host cells is still under-investigated. EXPERIMENTS: This work focuses on assessing changes in the acyl chain length of CH3(CH2)n-2CO-KKKIII-NH2 (n = 10, 12 and 14, K = lysine, I = isoleucine, denoted as CnKI3) on the antimicrobial potency and cytotoxicity by combining biological assays with physical measurements. Aggregation properties were characterized by changes in critical aggregation concentration (CAC) from surface tension measurements. Antimicrobial susceptibility tests, cytotoxic MTT assays, haemolytic tests, and dynamic bactericidal experiments were employed to reveal their bioactive potency toward different types of cells. To further investigate lipopeptides' underlying antimicrobial and cytotoxic mechanisms, lipid monolayer and lipid small unilamellar vesicle (SUV) models were established and biophysically characterized. FINDINGS: An increase in n led to the decrease in the CAC of CnKI3, showing a rising membrane-lytic power. Subsequent bioactive measurements revealed the optimal performance of C12KI3 from this series of lipopeptides. The selective membrane binding behaviour was well supported by neutron reflection data from charged lipid monolayer models, revealing membrane-supported nanostructures of ALPs. However, increased membrane-lytic actions in C14KI3 led to notably increased toxicity and reduced selectivity. On the other hand, C14KI3 can impose faster dynamic killing than natural lipopeptide polymyxin B, showing the distinct impact of the amphiphilic balance from the designed lipopeptide. In contrast, the distinctly weaker binding to zwitterionic membrane models (monolayers and SUVs) provided direct nanoscale structural evidence to the mildness of the designed ALPs on host cells. This work demonstrates the high selectivity and fast killing of rationally designed short ALPs to microbes via in-membrane nanostructuring.

In-Membrane Nanostructuring of Cationic Amphiphiles Affects Their Antimicrobial Efficacy and Cytotoxicity: A Comparison Study between a De Novo Antimicrobial Lipopeptide and Traditional Biocides.

Cationic biocides have been widely used as active ingredients in personal care and healthcare products for infection control and wound treatment for a long time, but there are concerns over their cytotoxicity and antimicrobial resistance. Designed lipopeptides are potential candidates for alleviating these issues because of their mildness to mammalian host cells and their high efficacy against pathogenic microbial membranes. In this study, antimicrobial and cytotoxic properties of a de novo designed lipopeptide, CH3(CH2)12CO-Lys-Lys-Gly-Gly-Ile-Ile-NH2 (C14KKGGII), were assessed against that of two traditional cationic biocides CnTAB (n = 12 and 14), with different critical aggregation concentrations (CACs). C14KKGGII was shown to be more potent against both bacteria and fungi but milder to fibroblast host cells than the two biocides. Biophysical measurements mimicking the main features of microbial and host cell membranes were obtained for both lipid monolayer models using neutron reflection and small unilamellar vesicles (SUVs) using fluorescein leakage and zeta potential changes. The results revealed selective binding to anionic lipid membranes from the lipopeptide and in-membrane nanostructuring that is distinctly different from the co-assembly of the conventional CnTAB. Furthermore, CnTAB binding to the model membranes showed low selectivity, and its high cytotoxicity could be attributed to both membrane lysis and chemical toxicity. This work demonstrates the advantages of the lipopeptides and their potential for further development toward clinical application.

Structural features of interfacially adsorbed acyl-l-carnitines.

HYPOTHESIS: Acyl-l-carnitines (CnLCs) are potentially important as biosurfactants in drug delivery and tissue engineering due to their good biocompatibility. However, little is currently known about the basic interfacial behavior underlying their technological applications. Following our previous characterization of their solution aggregation and adsorption at the air/water interface, this work examines how they adsorb at the hydrophilic solid/liquid interface. EXPERIMENTS: As the SiO2/water interface has served as the model substrate for many interfacial adsorption studies, so it has been used in this work as the solid substrate to facilitate dynamic adsorption by spectroscopic ellipsometry (SE) and structural determination of the adsorbed layers by neutron reflection (NR) under different conditions at the SiO2/water interface from a group of CnLC (n = 12, 14, and 16). FINDINGS: CnLC surfactants are zwitterionic at neutral pH. They reached saturated adsorption above their critical micellar concentrations (CMCs) and formed a sandwich bilayer with a head-tail-head structure at the hydrophilic SiO2/water interface. The total thicknesses of the adsorbed layers at CMC were found to be 33 ± 2, 35 ± 2, and 37 ± 2 Å for C12LC, C14LC, and C16LC, respectively, with their inner and outer head layers remaining similar but the thickness of the interdigitated middle layer increasing with acyl chain length. As the solution becomes acidic, the carboxyl groups become protonated and the l-carnitine heads are net positively charged, resulting in increased repulsion between the head groups. In this situation, the CnLC surfactants are adsorbed as distinct aggregates to reduce repulsive interaction, resulting in reduced surfactant volume fraction and layer thickness. However, a high ionic strength can screen the repulsive interaction and enhance the adsorbed amount, effectively diminishing the impact of pH. This information provides a useful basis for exploring the technological applications of CnLCs involving a solid substrate.

How do chain lengths of acyl-l-carnitines affect their surface adsorption and solution aggregation?

HYPOTHESIS: l-carnitines in our body systems can be readily converted into acyl-l-carnitines which have a prominent place in cellular energy generation by supporting the transport of long-chain fatty acids into mitochondria. As biocompatible surfactants, acyl-l-carnitines have potential to be useful in technical, personal care and healthcare applications. However, the lack of understanding of the effects of their molecular structures on their physical properties has constrained their potential use. EXPERIMENTS: This work reports the study of the influence of the acyl chain lengths of acyl-l-carnitines (CnLC) on solubility, surface adsorption and aggregation. Critical micellar concentrations (CMCs) of CnLC were determined by surface tension measurements. Neutron reflection (NR) was used to further examine the structure and composition of the adsorbed CnLC layer. The structural changes of the micellar aggregates under different concentrations of CnLC, pH and ionic strength were determined by dynamic light scattering (DLS) and small angle neutron scattering (SANS). FINDINGS: C12LC is fully soluble over a wide temperature and concentration range. There is however a strong decline of solubility with increasing acyl chain length. The adsorption and aggregation behavior of C14LC was therefore studied at 30 °C and C16LC at 45 °C. The solubility boundaries displayed distinct hysteresis with respect to heating and cooling. The CMCs of C12LC, C14LC and C16LC at pH 7 were 1.1 ± 0.1, 0.10 ± 0.02 and 0.010 ± 0.005 mM, respectively, with the limiting values of the area per molecule at the CMC being 45.4 ± 2, 47.5 ± 2 and 48.8 ± 2 Å2 and the thicknesses of the adsorbed CnLC layers at the air/water interface increasing from 21.5 ± 2 to 22.6 ± 2 to 24.2 ± 2 Å, respectively. All three surfactants formed core-shell spherical micelles with comparable dimensional parameters apart from an increase in core radius with acyl chain length. This study outlines the effects of acyl chain length on the physicochemical properties of CnLCs under different environmental conditions, serving as a useful basis for developing their potential applications.

Structural Disruptions of the Outer Membranes of Gram-Negative Bacteria by Rationally Designed Amphiphilic Antimicrobial Peptides.

Gram-negative bacteria are covered by both an inner cytoplasmic membrane (IM) and an outer membrane (OM). Antimicrobial peptides (AMPs) must first permeate through the OM and cell wall before attacking the IM to cause cytoplasmic leakage and kill the bacteria. The bacterial OM is an asymmetric bilayer with the outer leaflet primarily composed of lipopolysaccharides (LPSs) and the inner leaflet composed of phospholipids (PLs). Two cationic α-helical AMPs were designed to target Gram-negative bacteria, a full peptide G(IIKK)3I-NH2 (G3), and a hydrophobic lipopeptide C8-G(IIKK)2I-NH2 (C8G2, with C8 denoting the octanoyl chain). LPS dominates OM functions as the first line of defense against antibiotics, thereby reducing drug susceptibility. This work explores how the two AMPs interact with LPS through several carefully chosen OM models that facilitated measurements from solid-state nuclear magnetic resonance (ss-NMR), small-angle neutron scattering (SANS), and neutron reflectivity (NR). The results revealed that G3 molecules bound preferably to the LPS head region and functioned as bridge molecules to reassemble the dislocated lipids into bilayer stacks. In contrast, C8G2 lipopeptides could quickly penetrate into the central region of the OM to cause direct removal of some membrane lipids. Different structural disruptions implicated different antimicrobial efficacies from these AMPs. The demonstration of the structural features underlying different susceptibilities of the OM to AMPs offers a useful route for the future development of strain-specific AMPs against antimicrobial-resistant pathogens.

Acute pancreatitis with pulmonary embolism: A case report.

BACKGROUND: Acute pancreatitis (AP) is a common critical disease of the digestive system that is often associated with multiple complications. Vascular complications are relatively rare and are one of the causes of death. AP complicated with pulmonary embolism (PE) is even rarer, and there are no reports of AP complicated with PE in elderly patients. CASE SUMMARY: We describe a rare case of AP complicated with PE and review the literature. A 68-year-old woman was diagnosed with AP due to widespread abdominal pain. During the course of treatment, the patient had shortness of breath and progressively worsening dyspnea without chest pain or hemoptysis with a progressive increase in D-dimer and fibrin degradation product. Respiratory failure and right heart failure occurred, and refractory hypoxemia remained after mechanical ventilation. Plain chest computed tomography revealed a small amount of left pleural effusion and external pressure atelectasis in the lower lobe of the left lung but no findings that could lead to refractory hypoxemia. Color Doppler ultrasound indicated pulmonary hypertension and extensive venous thrombosis in the lower extremities. Chest computed tomography angiography finally suggested pulmonary thromboembolism. The patient's dyspnea symptoms disappeared after anticoagulation treatment. CONCLUSION: During the diagnosis and treatment of AP, it is necessary to dynamically monitor D-dimer and consider PE.

Surface adsorption and solution aggregation of a novel lauroyl-l-carnitine surfactant.

HYPOTHESIS: l-carnitine plays a crucial role in the cellular production of energy by transporting fatty acids into mitochondria. Acylated l-carnitines are amphiphilic and if appropriate physical properties were demonstrated, they could replace many currently used surfactants with improved biocompatibility and health benefits. EXPERIMENTS: This work evaluated the surface adsorption of lauroyl-l-carnitine (C12LC) and its aggregation behavior. The size and shape of the aggregates of C12LC surfactant were studied at different temperatures, concentrations, pH and ionic strength by dynamic light scattering (DLS) and small-angle neutron scattering (SANS). Surface tension measurements were carried out to determine the critical micellar concentration (CMC) of C12LC. Combining with the Gibbs equation, the surface excess at different concentrations could be determined. Neutron reflection (NR) was used to determine the structure of the adsorbed layer at the air/water interface with the help of isotopic contrast variations. FINDINGS: At pH 7, the limiting area per molecule (ACMC) of the zwitterionic C12LC adsorbed layer at the air/water interface was found to be 46 Å2 from surface tension and neutron reflection, smaller than the values of C12PC, C12E5, DTAB, C12C4betaine and C12C8betaine but close to that of SDS. A pronounced surface tension minimum at pH 2 at the low ionic strength was linked to a minimum value of area per molecule of about 30 Å2, indicating the competitive adsorption from traces of lauric acid produced by hydrolysis of C12LC. As the concentration increased, area per molecule reached a plateau of 37-39 Å2, indicating the dissolution of the more surface-active lauric acid into the micelles of C12LC. DLS and SANS showed that the size and shape of micelles had little response to temperature, concentration, ionic strength or pH. The SANS profiles measured under 3 isotopic contrasts could be well fitted by the core-shell model, giving a spherical core radius of 15.7 Å and a shell thickness of 10.5 Å. The decrease of pH led to more protonated carboxyl groups and more positively charged micelles, but the micellar structures remained unchanged, in spite of their stronger interaction. These features make C12LC potentially attractive as a solubilizing agent.

How do Self-Assembling Antimicrobial Lipopeptides Kill Bacteria?

Antimicrobial peptides are promising alternatives to traditional antibiotics. A group of self-assembling lipopeptides was formed by attaching an acyl chain to the N-terminus of α-helix-forming peptides with the sequence Cx-G(IIKK)yI-NH2 (CxGy, x = 4-12 and y = 2). CxGy self-assemble into nanofibers above their critical aggregation concentrations (CACs). With increasing x, the CACs decrease and the hydrophobic interactions increase, promoting secondary structure transitions within the nanofibers. Antimicrobial activity, determined by the minimum inhibition concentration (MIC), also decreases with increasing x, but the MICs are significantly smaller than the CACs, suggesting effective bacterial membrane-disrupting power. Unlike conventional antibiotics, both C8G2 and C12G2 can kill Staphylococcus aureus and Escherichia coli after only minutes of exposure under the concentrations studied. C12G2 nanofibers have considerably faster killing dynamics and lower cytotoxicity than their nonaggregated monomers. Antimicrobial activity of peptide aggregates has, to date, been underexploited, and it is found to be a very promising mechanism for peptide design. Detailed evidence for the molecular mechanisms involved is provided, based on superresolution fluorescence microscopy, solid-state nuclear magnetic resonance, atomic force microscopy, neutron scattering/reflectivity, circular dichroism, and Brewster angle microscopy.

Aggregated Amphiphilic Antimicrobial Peptides Embedded in Bacterial Membranes.

Molecular dynamics (MD) simulations, stochastic optical reconstruction microscopy (STORM), and neutron reflection (NR) were combined to explore how antimicrobial peptides (AMPs) can be designed to promote the formation of nanoaggregates in bacterial membranes and impose effective bactericidal actions. Changes in the hydrophobicity of the designed AMPs were found to have a strong influence on their bactericidal potency and cytotoxicity. G(IIKK)3I-NH2 (G3) achieved low minimum inhibition concentrations (MICs) and effective dynamic kills against both antibiotic-resistant and -susceptible bacteria. However, a G3 derivative with weaker hydrophobicity, KI(KKII)2I-NH2 (KI), exhibited considerably lower membrane-lytic activity. In contrast, the more hydrophobic G(ILKK)3L-NH2 (GL) peptide achieved MICs similar to those observed for G3 but with worsened hemolysis. Both the model membranes studied by Brewster angle microscopy, zeta potential measurements, and NR and the real bacterial membranes examined with direct STORM contained membrane-inserted peptide aggregates upon AMP exposure. These structural features were well supported by MD simulations. By revealing how AMPs self-assemble in microbial membranes, this work provides important insights into AMP mechanistic actions and allows further fine-tuning of antimicrobial potency and cytotoxicity.

Hydrophobic Control of the Bioactivity and Cytotoxicity of de Novo-Designed Antimicrobial Peptides.

Antimicrobial peptides (AMPs) can target bacterial membranes and kill bacteria through membrane structural damage and cytoplasmic leakage. A group of surfactant-like cationic AMPs was developed from substitutions to selective amino acids in the general formula of G(IIKK)3I-NH2, (called G3, a de novo AMP), to explore the correlation between AMP hydrophobicity and bioactivity. A threshold surface pressure over 12 mN/m was required to cause measurable antimicrobial activity and this corresponded to a critical AMP concentration. Greater surface activity exhibited stronger antimicrobial activity but had the drawback of worsening hemolytic activity. Small unilamellar vesicles (SUVs) with specific lipid compositions were used to model bacterial and host mammalian cell membranes by mimicking the main structural determinants of the charge and composition. Leakage from the SUVs of encapsulated carboxyfluorescein measured by fluorescence spectroscopy indicated a negative correlation between hydrophobicity and model membrane selectivity, consistent with measurements of the zeta potential that demonstrated the extent of AMP binding onto model SUV lipid bilayers. Experiments with model lipid membranes thus explained the trend of minimum inhibitory concentrations and selectivity measured from real cell systems and demonstrated the dominant influence of hydrophobicity. This work provides useful guidance for the improvement of the potency of AMPs via structural design, whilst taking due consideration of cytotoxicity.

Integrated mapping of pharmacokinetics and pharmacodynamics in a patient-derived xenograft model of glioblastoma.

Therapeutic options for the treatment of glioblastoma remain inadequate despite concerted research efforts in drug development. Therapeutic failure can result from poor permeability of the blood-brain barrier, heterogeneous drug distribution, and development of resistance. Elucidation of relationships among such parameters could enable the development of predictive models of drug response in patients and inform drug development. Complementary analyses were applied to a glioblastoma patient-derived xenograft model in order to quantitatively map distribution and resulting cellular response to the EGFR inhibitor erlotinib. Mass spectrometry images of erlotinib were registered to histology and magnetic resonance images in order to correlate drug distribution with tumor characteristics. Phosphoproteomics and immunohistochemistry were used to assess protein signaling in response to drug, and integrated with transcriptional response using mRNA sequencing. This comprehensive dataset provides simultaneous insight into pharmacokinetics and pharmacodynamics and indicates that erlotinib delivery to intracranial tumors is insufficient to inhibit EGFR tyrosine kinase signaling.

Simultaneous two-color stimulated Raman scattering microscopy by adding a fiber amplifier to a 2 ps OPO-based SRS microscope.

Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technique. Two-color imaging is often necessary to determine the distribution of chemical species in SRS microscopy. Current multi-color SRS imaging methods involve complicated instrumentation or longer data acquisition time or are limited to transmission imaging. In this Letter, we show that by adding a simple fiber amplifier to a 2 ps laser source and optical-parametric-oscillator-based SRS setup, one can achieve simultaneous two-color or frequency modulation SRS microscopy. The fiber amplifier can generate a wavelength tunable laser of ±10 nm around the Stokes laser wavelength at 1031 nm with average power greater than 200 mW. In vivo and ex vivo lipid-protein imaging of mouse brain and skin is demonstrated. To further demonstrate the potential of this technique in high-speed in vivo imaging, white blood cells in a blood stream are imaged in a live mouse.

Monitoring peripheral nerve degeneration in ALS by label-free stimulated Raman scattering imaging.

The study of amyotrophic lateral sclerosis (ALS) and potential interventions would be facilitated if motor axon degeneration could be more readily visualized. Here we demonstrate that stimulated Raman scattering (SRS) microscopy could be used to sensitively monitor peripheral nerve degeneration in ALS mouse models and ALS autopsy materials. Three-dimensional imaging of pre-symptomatic SOD1 mouse models and data processing by a correlation-based algorithm revealed that significant degeneration of peripheral nerves could be detected coincidentally with the earliest detectable signs of muscle denervation and preceded physiologically measurable motor function decline. We also found that peripheral degeneration was an early event in FUS as well as C9ORF72 repeat expansion models of ALS, and that serial imaging allowed long-term observation of disease progression and drug effects in living animals. Our study demonstrates that SRS imaging is a sensitive and quantitative means of measuring disease progression, greatly facilitating future studies of disease mechanisms and candidate therapeutics.

Label-Free Neurosurgical Pathology with Stimulated Raman Imaging.

The goal of brain tumor surgery is to maximize tumor removal without injuring critical brain structures. Achieving this goal is challenging as it can be difficult to distinguish tumor from nontumor tissue. While standard histopathology provides information that could assist tumor delineation, it cannot be performed iteratively during surgery as freezing, sectioning, and staining of the tissue require too much time. Stimulated Raman scattering (SRS) microscopy is a powerful label-free chemical imaging technology that enables rapid mapping of lipids and proteins within a fresh specimen. This information can be rendered into pathology-like images. Although this approach has been used to assess the density of glioma cells in murine orthotopic xenografts models and human brain tumors, tissue heterogeneity in clinical brain tumors has not yet been fully evaluated with SRS imaging. Here we profile 41 specimens resected from 12 patients with a range of brain tumors. By evaluating large-scale stimulated Raman imaging data and correlating this data with current clinical gold standard of histopathology for 4,422 fields of view, we capture many essential diagnostic hallmarks for glioma classification. Notably, in fresh tumor samples, we observe additional features, not seen by conventional methods, including extensive lipid droplets within glioma cells, collagen deposition in gliosarcoma, and irregularity and disruption of myelinated fibers in areas infiltrated by oligodendroglioma cells. The data are freely available in a public resource to foster diagnostic training and to permit additional interrogation. Our work establishes the methodology and provides a significant collection of reference images for label-free neurosurgical pathology. Cancer Res; 76(12); 3451-62. ©2016 AACR.

Optical technologies for intraoperative neurosurgical guidance.

Biomedical optics is a broadly interdisciplinary field at the interface of optical engineering, biophysics, computer science, medicine, biology, and chemistry, helping us understand light-tissue interactions to create applications with diagnostic and therapeutic value in medicine. Implementation of biomedical optics tools and principles has had a notable scientific and clinical resurgence in recent years in the neurosurgical community. This is in great part due to work in fluorescence-guided surgery of brain tumors leading to reports of significant improvement in maximizing the rates of gross-total resection. Multiple additional optical technologies have been implemented clinically, including diffuse reflectance spectroscopy and imaging, optical coherence tomography, Raman spectroscopy and imaging, and advanced quantitative methods, including quantitative fluorescence and lifetime imaging. Here we present a clinically relevant and technologically informed overview and discussion of some of the major clinical implementations of optical technologies as intraoperative guidance tools in neurosurgery.

Patterns and possible mechanisms of soil CO2 uptake in sandy soil.

It has been reported that soils in drylands can absorb CO2, although the patterns and mechanisms of such a process remain under debate. To address this, we investigated the relationships between soil CO2 flux and meteorological factors and soil properties in Northwest China to reveal the reasons for "anomalous" soil CO2 flux in a desert ecosystem. Soil CO2 flux increased significantly and exponentially with surficial turbulence at the diel scale under dry conditions (P<0.05), whereas the relationship under wet conditions was insignificant. Furthermore, soil CO2 flux demonstrated remarkable negative correlation with soil air pressure (P<0.05) in both dry and wet conditions. Analysis considering Henry's Law indicated that soil water content was insufficient to dissolve the absorbed CO2 in dry conditions, but was sufficient in wet conditions. The concentration of soil HCO3(-) in the morning was higher than in the evening in dry conditions, but this pattern was reversed in wet conditions. These results imply that CO2 outgassing induced by turbulence, expansion of soil air, CO2 effusion from soil water, and carbonate precipitation during daytime can explain the abiotic diurnal CO2 release. Moreover, CO2 pumping from the atmosphere into the soil, caused mainly by carbonate dissolution, can account for nocturnal CO2 absorption in dry conditions. The abiotic soil CO2 flux pattern (CO2 absorption throughout the diel cycle) in wet conditions can be attributed to downward mass flow of soil CO2 and intensified soil air shrinkage, CO2 dissolving in soil water, and carbonate dissolution. These results provide a basis for determining the location of abiotic fixed carbon within soils in desert ecosystems.

Label-free DNA imaging in vivo with stimulated Raman scattering microscopy.

Label-free DNA imaging is highly desirable in biology and medicine to perform live imaging without affecting cell function and to obtain instant histological tissue examination during surgical procedures. Here we show a label-free DNA imaging method with stimulated Raman scattering (SRS) microscopy for visualization of the cell nuclei in live animals and intact fresh human tissues with subcellular resolution. Relying on the distinct Raman spectral features of the carbon-hydrogen bonds in DNA, the distribution of DNA is retrieved from the strong background of proteins and lipids by linear decomposition of SRS images at three optimally selected Raman shifts. Based on changes on DNA condensation in the nucleus, we were able to capture chromosome dynamics during cell division both in vitro and in vivo. We tracked mouse skin cell proliferation, induced by drug treatment, through in vivo counting of the mitotic rate. Furthermore, we demonstrated a label-free histology method for human skin cancer diagnosis that provides comparable results to other conventional tissue staining methods such as H&E. Our approach exhibits higher sensitivity than SRS imaging of DNA in the fingerprint spectral region. Compared with spontaneous Raman imaging of DNA, our approach is three orders of magnitude faster, allowing both chromatin dynamic studies and label-free optical histology in real time.

Effect of vegetation rehabilitation on soil carbon and its fractions in Mu Us Desert, northwest China.

Although vegetation rehabilitation on semi-arid and arid regions may enhance soil carbon sequestration, its effects on soil carbon fractions remain uncertain. We carried out a study after planting Artemisia ordosica (AO, 17 years), Astragalus mongolicum (AM, 5 years), and Salix psammophila (SP, 16 years) on shifting sand land (SL) in the Mu Us Desert, northwest China. We measured total soil carbon (TSC) and its components, soil inorganic carbon (SIC) and soil organic carbon (SOC), as well as the light and heavy fractions within soil organic carbon (LF-SOC and HF-SOC), under the SL and shrublands at depths of 100 cm. TSC stock under SL was 27.6 Mg ha(-1), and vegetation rehabilitation remarkably elevated it by 40.6 Mgha(-1), 4.5 Mgha(-1), and 14.1 Mgha(-1) under AO, AM and SP land, respectively. Among the newly formed TSC under the three shrublands, SIC, LF-SOC and HF-SOC accounted for 75.0%, 10.7% and 13.1% for AO, respectively; they made up 37.0%, 50.7% and 10.6% for AM, respectively; they occupied 68.6%, 18.8% and 10.0% for SP, respectively. The accumulation rates of TSC within 0-100 cm reached 238.6 g m(-2) y(-1), 89.9 g m(-2) y(-1) and 87.9 g m(-2) y(-1) under AO, AM and SP land, respectively. The present study proved that the accumulation of SIC considerably contributed to soil carbon sequestration, and vegetation rehabilitation on shifting sand land has a great potential for soil carbon sequestration.

Influence of disturbance on soil respiration in biologically crusted soil during the dry season.

Soil respiration (Rs) is a major pathway for carbon cycling and is a complex process involving abiotic and biotic factors. Biological soil crusts (BSCs) are a key biotic component of desert ecosystems worldwide. In desert ecosystems, soils are protected from surface disturbance by BSCs, but it is unknown whether Rs is affected by disturbance of this crust layer. We measured Rs in three types of disturbed and undisturbed crusted soils (algae, lichen, and moss), as well as bare land from April to August, 2010, in Mu Us desert, northwest China. Rs was similar among undisturbed soils but increased significantly in disturbed moss and algae crusted soils. The variation of Rs in undisturbed and disturbed soil was related to soil bulk density. Disturbance also led to changes in soil organic carbon and fine particles contents, including declines of 60-70% in surface soil C and N, relative to predisturbance values. Once BSCs were disturbed, Q 10 increased. Our findings indicate that a loss of BSCs cover will lead to greater soil C loss through respiration. Given these results, understanding the disturbance sensitivity impact on Rs could be helpful to modify soil management practices which promote carbon sequestration.