Journal: Nature Communications

Article Title: Translation control during prolonged mTORC1 inhibition mediated by 4E-BP3

doi: 10.1038/ncomms11776

Figure Lengend Snippet: ( a ) Sequence (−163 to +59) in the EIF4EBP3 promoter region. Position +1 indicates the information of putative transcription start site (the DBTSS ( http://dbtss.hgc.jp )). Protein coding region is shown by red characters. ( b ) E-box mutation reduces EIF4EBP3 promoter activity. E-box sequences 5′-CAGCTG CACGTG-3′ were changed to 5′-GTCCTG GTGGTG-3′. MiaPaCa-2 cells were transiently transfected with p4E-BP3pro-Luc (−163 to +59) plasmid or its E-boxes mutant and pRL-TK reporter plasmid as a control. After 24 h, cells were treated with 100 nM of INK1341 for 24 h. Relative luciferase activity was measured using Dual-Luciferase Assay kit. Error bars indicate±s.d. ( n =3). ** P <0.01. Statistical significance was determined using two-way analysis of variance (ANOVA). A representative result of three independent experiments is shown. ( c ) TFE3 and TFEB expression in three cell lines. Each mRNA expression was analysed by RT–PCR. ( d ) MiaPaCa-2 cells were transiently transfected with the indicated amount of TFE3 (HA-tag) plasmid. Detection of the proteins was performed by immunoblotting. A representative result of two independent experiments is shown. ( e ) MiaPaCa-2 cells were transiently transfected with the indicated amount of TFE3 (HA-tag) plasmid together with reporter plasmids as shown in b . After 48 h, Relative luciferase activity was measured using Dual-Luciferase Assay kit. Error bars indicate±s.d. ( n =3). ** P <0.01. Statistical significance was determined using two-way ANOVA. A representative result of three independent experiments is shown.

Article Snippet: Antibody against human TFE3 (A302-622A) was purchased from Bethyl (Montgomery, TX).

Techniques: Sequencing, Mutagenesis, Activity Assay, Transfection, Plasmid Preparation, Luciferase, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Nature Communications

Article Title: Translation control during prolonged mTORC1 inhibition mediated by 4E-BP3

doi: 10.1038/ncomms11776

Figure Lengend Snippet: ( a ) MiaPaCa-2 cells were treated with 100 nM of INK1341 for 1 h. The cells were fixed and endogenous TFE3 protein in cells was stained with anti-TFE3 antibody and Alexa-Fluor 488. Scale bar, 10 μm. ( b ) MiaPaCa-2 cells were treated with 100 nM of INK1341 for 1 h. Cells were lysed with a Dounce homogenizer. Nuclear and cytoplasmic fractions were separated by centrifugation. Protein levels were determined by immunoblotting. Arrow shows phosphorylated or dephosphorylated form (arrow with star). ( c ) Cells were treated with 100 nM of INK1341 for 1 h. Binding of TFE3 to the E-box region in EIF4EBP3 promoter was analysed by chromatin immunoprecipitation assay. ( d , e ) MiaPaCa-2 cells were transduced with scrambled (sh-Scr) or TFE3 shRNA (sh-TFE3). Protein and mRNA levels were determined by immunoblotting and real-time PCR analysis, respectively. mRNA levels were normalized against β-Actin. Error bars indicate±s.d. ( n =3). * P <0.05 and ** P <0.01. Statistical significance was determined using two-way analysis of variance (ANOVA). ( f ) MiaPaCa-2 cells expressing scrambled or TFE3 shRNA were transiently transfected with reporter plasmids (p4E-BP3pro-Luc (−163 to +59) plasmid and pRL-TK). After 24 h, the cells were treated with rapamycin (100 nM), INK1341 (100 nM) or PP242 (1 μM) for 24 h and then relative luciferase activity was determined as shown in b . Error bars indicate±s.d. ( n =3). * P <0.05 and ** P <0.01. Statistical significance was determined using two-way ANOVA. A representative result of three ( a , b ) or two independent experiments ( c – f ) is shown.

Article Snippet: Antibody against human TFE3 (A302-622A) was purchased from Bethyl (Montgomery, TX).

Techniques: Staining, Centrifugation, Western Blot, Binding Assay, Chromatin Immunoprecipitation, Transduction, shRNA, Real-time Polymerase Chain Reaction, Expressing, Transfection, Plasmid Preparation, Luciferase, Activity Assay

Journal: bioRxiv

Article Title: The Fraser complex interconnects tissue layers to support basal epidermis and osteoblast integrated morphogenesis underlying fin skeletal patterning

doi: 10.1101/2023.07.08.548238

Figure Lengend Snippet: (A) Whole-mount imaged adult zebrafish representing three homozygous fras1 −/− mutants and a heterozygous sibling. fras1 −/− fish are variably runted with gross abnormalities including caudal fin pigment pattern defects (black arrows), misshapen anal fins (black arrowheads), and lower jaw protrusions (black asterisk). (B-F) Representative Alizarin Red-stained caudal fins of (B) control and (C-F) fras1 −/− adult siblings. Unbranched rays are shaded green as a visual aid. Dorsal and ventral procurrent rays (dPR and vPR) are labeled with cyan arrows. Asymmetric fins (D, E) are indicated by white “asym.” brackets. (C’-F’) Various skeletal patterning abnormalities highlighted by zoomed in regions shown by yellow dashed boxes in (C-F) and noted below. (C’) Fused peripheral rays (yellow arrow), ectopic branching (yellow arrowhead), and fracture scar (yellow open arrowhead). (D’) Three unbranched rays (green arrowheads) followed by a branched ray followed by an ectopic, partially branched peripheral ray (yellow arrowhead). (E’) Uneven branch points (white connected dots), poorly articulated joints (yellow arrow), wavy misaligned rays (yellow bracket). (F’) Numerous fused rays (yellow connected dots) and isolated ray fusion (white arrowhead). (G-I) Individual data point plots showing control and fras1 −/− adult caudal fin (G) principal ray number, (H) procurrent ray number, and (I) fin ray branching expressed as a fraction of total principal rays. Means are shown.

Article Snippet: An anti-FRAS1 human antibody with 76% immunogen identity with zebrafish Fras1 including part of a highly conserved Calx-beta domain (Bethyl Labs, Cat #A305-622A) and anti-FREM2 human antibody with 77% immunogen identity with zebrafish Frem2a/b (Invitrogen PA5-55986) were used.

Techniques: Staining, Labeling, Isolation

Journal: bioRxiv

Article Title: The Fraser complex interconnects tissue layers to support basal epidermis and osteoblast integrated morphogenesis underlying fin skeletal patterning

doi: 10.1101/2023.07.08.548238

Figure Lengend Snippet: (A) Fin ray scoring for all 7 zebrafish adult fins for n = 5 fras1 +/− heterozygote controls and n = 8 fras1 −/− homozygote mutants. The paired pectoral and pelvic fins have two data points per fish. (B) Ratio of branched fin rays over total fin rays per fin. The paired pectoral and pelvic fins have two data points per fish. (C) Pectoral fin ray number and (D) Pelvic fin ray number for right (R) and left (L) fins with data points for individual control and fras1 −/− fish in matching blue or red spectrum colors, respectively. Means are shown. (E-R) Representative Alizarin Red-stained adult caudal fins overlayed with brightfield for (E, F, I, J, M, O, Q) controls and (G, H, K, L, N, P, R) fras1 −/− mutants. (E-H) Pectoral and (I-L) pelvic fins are from the same animal to highlight left-right asymmetry in fras1 −/− mutants. Number of rays, branched rays, and scale bars are as marked.

Article Snippet: An anti-FRAS1 human antibody with 76% immunogen identity with zebrafish Fras1 including part of a highly conserved Calx-beta domain (Bethyl Labs, Cat #A305-622A) and anti-FREM2 human antibody with 77% immunogen identity with zebrafish Frem2a/b (Invitrogen PA5-55986) were used.

Techniques: Staining

Journal: bioRxiv

Article Title: The Fraser complex interconnects tissue layers to support basal epidermis and osteoblast integrated morphogenesis underlying fin skeletal patterning

doi: 10.1101/2023.07.08.548238

Figure Lengend Snippet: (A-H) Representative whole mount imaging of Alcian Blue (cartilage) and Alizarin Red (calcified bone) stained zebrafish of indicated fras1 genotypes and ages. (A) Late larval fras1 +/− control caudal fin. H1, hypural 1 and H5, hypural 5. Magenta brackets denote joint segmentation. (B) Trunk region of the same control larvae. Dorsal and anal fin radials are marked by white brackets and rays are marked by black dashed arrows. (C-H) Images of sibling fras1 −/− late larvae age-matched (C, D) or size-matched (E-H) to control for frequent developmental delays, with highlighted defects described below. (C, E, G) Caudal fin endoskeletons (blue) are disorganized, often with a sixth hypural (H6, yellow), fused elements (yellow dashed lines), and ectopic cartilaginous structures (yellow arrows). (E) Caudal fin dermal rays (magenta) are variably misaligned ( C, G ; yellow brackets) and (E, G) have delayed joint development (magenta open arrowheads) even when size-matched to (A) younger controls. (D, F, H) Dorsal and anal fin endoskeletons (blue) have variably mispatterned radials (green asterisks), with dorsal fins often having extra radials (green arrows). Scale bars are 1 mm. (I) Data point plots scoring indicated fin skeletal elements or defects in fras1 −/− mutants and control siblings. Means are shown.

Article Snippet: An anti-FRAS1 human antibody with 76% immunogen identity with zebrafish Fras1 including part of a highly conserved Calx-beta domain (Bethyl Labs, Cat #A305-622A) and anti-FREM2 human antibody with 77% immunogen identity with zebrafish Frem2a/b (Invitrogen PA5-55986) were used.

Techniques: Imaging, Staining

Journal: bioRxiv

Article Title: The Fraser complex interconnects tissue layers to support basal epidermis and osteoblast integrated morphogenesis underlying fin skeletal patterning

doi: 10.1101/2023.07.08.548238

Figure Lengend Snippet: (A-D) Images of representative fras1 +/− heterozygote control adult caudal fins before injury (A, uninjured) and at 3 (B) , 7 (C) , and 28 (D) days post amputation (dpa). (B’, C’) Zoom insets of the yellow boxed regions in B and C. (B’) Black arrows (A) indicate the longest principal ray and white arrows indicate the shortest. Magenta dashed line denotes the approximate boundary between the outgrowing ray field and distal epidermis. White dashed lines indicate the plane of amputation. (E-P) Three representative fras1 −/− homozygote siblings at the same regenerative time points as (A-D) highlighting defects noted below. At 3 dpa, mutants exhibit blistering (cyan asterisk; F’, N’ ) and extended epidermal tissue distal to the ray field (magenta arrowheads; F’, J’, N’ ). (G, G’, K, K’, O, O’) Blistering subsides by 7 dpa. (H-P) Fully regenerated mutant fins have (H) fewer branched rays, (L) no branched rays, or more equal ray length ratios (i.e., less pronounced V-shape) (C) compared to their uninjured states. Scale bar lengths are indicated. (Q-S) Individual data point plots comparing fin shape as well as ray length and branching restoration upon regeneration between control and fras1 −/− animals. (Q) Ray length ratios between the longest and shortest principal rays before injury (solid circles) and at 28 dpa (unfilled circles). Uninjured and regenerated ray length ratios are unchanged within genotypes (control P = 0.92, fras1 −/− P=0.22); paired t-tests) but are significantly different between genotypes (P <0.0001 for uninjured and regenerated fins; unpaired t-tests). (R) The fraction of branched over total principal rays, with connecting lines indicating the same fish assessed before injury and at 28 dpa. Ray branching frequency decreased after regeneration in all fras1 −/− animals (P = 0.012; paired t-test) but no control animals (P = 1). (S) The fraction of regenerated to original ray length for the longest and shortest principal rays and the ratio of the two values. Means and SD are shown.

Article Snippet: An anti-FRAS1 human antibody with 76% immunogen identity with zebrafish Fras1 including part of a highly conserved Calx-beta domain (Bethyl Labs, Cat #A305-622A) and anti-FREM2 human antibody with 77% immunogen identity with zebrafish Frem2a/b (Invitrogen PA5-55986) were used.

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: The Fraser complex interconnects tissue layers to support basal epidermis and osteoblast integrated morphogenesis underlying fin skeletal patterning

doi: 10.1101/2023.07.08.548238

Figure Lengend Snippet: (A) Schematic of epithelial (blue cells) and mesenchymal (pink cells) tissue layers separated by basement membrane (BM, black lines). The Fraser Complex (FPC, multicolored) localizes to the innermost BM layer (sublamina densa, dashed black line) and comprises Fras1 (magenta), Frem1a/b (cyan), Frem2a/b (purple), and Frem3 (yellow). (B) Longitudinal section schematic of tissue organization in a 3-day post amputation (dpa) regenerating caudal fin. (C) Single cell RNA-Seq clustering of regenerating 3 and 7 dpa zebrafish fin tissue shows unique clusters for basal epidermis (blue), superficial epidermis (green), and mixed mesenchyme/osteoblast/fibroblast lineages (dark red, pink, yellow) in UMAP space. (D) Bubble plot of FPC transcript expression in the basal epidermis, superficial epidermis (sEp), and mixed mesenchyme/osteoblast/fibroblast (Mes/Ob/Fib) clusters. (E-G) 40x confocal maximum intensity projections of regenerating fin longitudinal sections stained as follows: (E) Fras1 immunostaining (green) at 4 dpa, (F) ViewRNA fluorescent in situ hybridization for frem2a (cyan) at 3 dpa, and (G) ViewRNA fluorescent in situ hybridization for frem2b (magenta) at 3 dpa. Hoechst-stained nuclei are grey. (E’-G’) Single channel fluorescence images of (E-G) . Scale bars are 50 μm.

Article Snippet: An anti-FRAS1 human antibody with 76% immunogen identity with zebrafish Fras1 including part of a highly conserved Calx-beta domain (Bethyl Labs, Cat #A305-622A) and anti-FREM2 human antibody with 77% immunogen identity with zebrafish Frem2a/b (Invitrogen PA5-55986) were used.

Techniques: RNA Sequencing Assay, Expressing, Staining, Immunostaining, In Situ Hybridization, Fluorescence

Journal: bioRxiv

Article Title: The Fraser complex interconnects tissue layers to support basal epidermis and osteoblast integrated morphogenesis underlying fin skeletal patterning

doi: 10.1101/2023.07.08.548238

Figure Lengend Snippet: (A) UMAP cluster analysis from single cell transcriptomes of 3- and 7-day post amputation (dpa) zebrafish fin tissue identifying unique clusters for basal epidermis (blue), superficial epidermis (green), and mixed blastemal mesenchyme / osteoblasts / fibroblasts (dark red, pink, yellow). (B-G) Individual UMAP plots for Fraser Complex components fras1 , frem1a , frem1b , frem2a , frem2b , and frem3 . fras1 is the most highly enriched basal epidermal marker of all captured transcripts.

Article Snippet: An anti-FRAS1 human antibody with 76% immunogen identity with zebrafish Fras1 including part of a highly conserved Calx-beta domain (Bethyl Labs, Cat #A305-622A) and anti-FREM2 human antibody with 77% immunogen identity with zebrafish Frem2a/b (Invitrogen PA5-55986) were used.

Techniques: Marker

Journal: bioRxiv

Article Title: The Fraser complex interconnects tissue layers to support basal epidermis and osteoblast integrated morphogenesis underlying fin skeletal patterning

doi: 10.1101/2023.07.08.548238

Figure Lengend Snippet: (A-D) Confocal images of immunofluorescent stained paraffin sections of 4 day post amputation (4 dpa) regenerating caudal fins. Hoechst (gray) stains nuclei and anti-Runx2 antibody staining (magenta) marks pre-osteoblasts. Asterisks indicate blood autofluorescence. (A-D) Maximum intensity projection 3-channel overlays. (A’-D’) Single channel showing Fras1 or Frem2 immunostaining (green). (A”, B”) Maximum intensity projection zoomed views of the yellow boxed regions in (A-B) . (E, F) Single optical slice zooms of the yellow boxed regions in (C-D) . (A, A’) Fras1 (green) in wildtype fins is strongly expressed in the distal basement membrane with diminishing expression extending proximally. (A”) Fras1 is expressed at low levels between Runx2+ pre-osteoblasts (yellow outline) and epidermal cells. Epid, epidermis; Mes., mesenchyme. The cyan dashed line indicates the approximate basement membrane position. (B-B”) Fras1 (green) is lost and pre-osteoblasts are disorganized in fras1 −/− mutants. (C, C’, C”) Frem2 (green) is expressed in the distal basement membrane (white arrow) and in the extracellular space around basal epidermal cells (bEp, white arrowheads, C” ). (D-D”) Frem2 expression (green) in fras1 −/− mutants is absent from distal basement membrane (yellow arrow) and the remaining protein appears intracellular within basal epidermal cells (yellow arrowheads). Scale bar lengths are indicated.

Article Snippet: An anti-FRAS1 human antibody with 76% immunogen identity with zebrafish Fras1 including part of a highly conserved Calx-beta domain (Bethyl Labs, Cat #A305-622A) and anti-FREM2 human antibody with 77% immunogen identity with zebrafish Frem2a/b (Invitrogen PA5-55986) were used.

Techniques: Staining, Immunostaining, Expressing

Journal: bioRxiv

Article Title: The Fraser complex interconnects tissue layers to support basal epidermis and osteoblast integrated morphogenesis underlying fin skeletal patterning

doi: 10.1101/2023.07.08.548238

Figure Lengend Snippet: (A-D) Whole mount images of regenerating caudal fins of (A-B) fras1 +/− control sibling and (C-D) fras1 −/− zebrafish carrying the shha:GFP reporter. (A) At 8 days post amputation (dpa), shha:GFP is expressed in basal epidermal cells overlying the distal outgrowth zone of each ray. (A’) Zoom of ventral ray regions showing GFP domain splitting (green arrows) associated with a branched ray (magenta dashed outline). (C, C’) A fras1 −/− fish at 8 dpa with split distal GFP domains ( C’ ; green arrows) even while the underlying rays fail to branch (magenta dashed outline). (B, D) Regeneration is complete at 30 dpa, with only residual distal GFP. The fras1 −/− animal (D) fails to regenerate branched rays. White dashed lines indicate the amputation site. (E-H) Whole mount images of regenerating caudal fins from ptch2:Kaede sibling zebrafish of indicated fras1 genotype. (E, G) 4 dpa fins imaged immediately after ptch2:Kaede is photoconverted from green to red (shown in magenta) fluorescence in the marked field (yellow dashed boxes, 0 hours-post-conversion hpc). (F, H) The same fins imaged 24 hours later (at 5 dpa, 24 hpc) showing newly produced green Kaede domains in both genotypes ( F”, H” ; white arrows). (E’-H’) Double fluorescent and (E”-H”) single fluorescence channels. Scale bar lengths are indicated.

Article Snippet: An anti-FRAS1 human antibody with 76% immunogen identity with zebrafish Fras1 including part of a highly conserved Calx-beta domain (Bethyl Labs, Cat #A305-622A) and anti-FREM2 human antibody with 77% immunogen identity with zebrafish Frem2a/b (Invitrogen PA5-55986) were used.

Techniques: Fluorescence, Produced

Journal: bioRxiv

Article Title: The Fraser complex interconnects tissue layers to support basal epidermis and osteoblast integrated morphogenesis underlying fin skeletal patterning

doi: 10.1101/2023.07.08.548238

Figure Lengend Snippet: (A, B) Whole mount caudal fin images of representative (A) shha:GFP control ( n = 4) and (B) shha:GFP; fras1 −/− ( n = 6) siblings at six weeks post fertilization (wpf). (A’, B’) Zoom insets of the dorsal tip of the caudal fins. Dorsal ray 2 (dashed white outline) is branched in (A’) but unbranched in (B’) . (C-F’) Confocal fluorescence images of the dorsal lobe of developing caudal fins from a ptch2:Kaede control and stage-matched fras1 −/− mutant sibling ( n = 3 controls, n = 8 mutants). (C, E) White outlined regions show complete conversion of Kaede from green to red emission at 0 hours-post-conversion (hpc) following UV light exposure (single channel panels in C’, E’ ). (D, F) At 24 hpc, the same fish express small domains of green Kaede protein (green arrows) newly produced in the photoconverted region (white lines), indicating active Shh/Smo signaling. Dashed gray lines indicate fin boundaries.

Article Snippet: An anti-FRAS1 human antibody with 76% immunogen identity with zebrafish Fras1 including part of a highly conserved Calx-beta domain (Bethyl Labs, Cat #A305-622A) and anti-FREM2 human antibody with 77% immunogen identity with zebrafish Frem2a/b (Invitrogen PA5-55986) were used.

Techniques: Fluorescence, Mutagenesis, Produced

Journal: bioRxiv

Article Title: The Fraser complex interconnects tissue layers to support basal epidermis and osteoblast integrated morphogenesis underlying fin skeletal patterning

doi: 10.1101/2023.07.08.548238

Figure Lengend Snippet: (A-H’) Hematoxylin and eosin-stained longitudinal sections of 4 day post amputation (4 dpa) regenerating caudal fins from fras1 +/− control and fras1 −/− adult zebrafish. (A-D) Brightfield images with hematoxylin-marked nuclei (purple) and eosin-marked cytoplasm and ECM (pink). Representative fras +/− control (A) and fras1 −/− fish ( B-D ) are shown. sEp, superficial epidermis; bEp, basal epidermis; Mes., mesenchyme. (E-H) Confocal optical slices showing fluorescent eosin signal from the samples in (A-D) . Green arrowheads indicate epidermal blistering. Pink arrowheads indicate mesenchymal blistering. Orange brackets indicate excess epidermal tissue. (E’-H’) Zoom regions of yellow dashed boxes in (E-H) with approximate boundaries of the distal-most forming basement membrane (BM; cyan dashed line) and basal epidermis (bEp; magenta dashed line). Scale bars are indicated.

Article Snippet: An anti-FRAS1 human antibody with 76% immunogen identity with zebrafish Fras1 including part of a highly conserved Calx-beta domain (Bethyl Labs, Cat #A305-622A) and anti-FREM2 human antibody with 77% immunogen identity with zebrafish Frem2a/b (Invitrogen PA5-55986) were used.

Techniques: Staining

Journal: bioRxiv

Article Title: The Fraser complex interconnects tissue layers to support basal epidermis and osteoblast integrated morphogenesis underlying fin skeletal patterning

doi: 10.1101/2023.07.08.548238

Figure Lengend Snippet: (A, B) Schematic of epithelial basal epidermis (blue cells) and mesenchymal pre-osteoblast (salmon cells) tissue organization separated by an immature basement membrane (BM, black dashed lines) in the distal developing or regenerating fin ray. (A) The Fraser Complex (FPC) adheres epidermal and mesenchymal tissue layers to support osteoblast organization and therefore skeletal patterning. (B) In fras1 −/− mutants, Fraser Complex components cannot translocate to the sublamina densa basement membrane layer and blisters form between tissue layers. (C) Wildtype developing caudal endoskeleton with blue and purple representing cartilage and calcified bone, respectively. Abbreviations: UST, urostyle; PL, pleurostyle; OPC, opisthural cartilage; H1-H5, hypurals 1-5; PH, parhypural; HS, hemal spine. (D) Example of a fras1 −/− caudal endoskeleton, including ectopic cartilaginous structures (yellow arrowheads), an additional hypural (H6, yellow), fusions between elements (yellow asterisk), and general poor articulation. (E) Wildtype caudal fin dermal skeleton (fin rays) anatomy. (F) Example of fras1 −/− fin ray patterning defects. Abnormalities include ray fusions (green arrowhead), absent branching (open green arrowhead), uneven joints (green brackets), precocious branching (green arrow), and wavy rays (green asterisk).

Article Snippet: An anti-FRAS1 human antibody with 76% immunogen identity with zebrafish Fras1 including part of a highly conserved Calx-beta domain (Bethyl Labs, Cat #A305-622A) and anti-FREM2 human antibody with 77% immunogen identity with zebrafish Frem2a/b (Invitrogen PA5-55986) were used.

Techniques:

Journal: Scientific Reports

Article Title: A non-invasive multipoint product temperature measurement for pharmaceutical lyophilization

doi: 10.1038/s41598-022-16073-x

Figure Lengend Snippet: Primary drying stage process parameters for recipe described in Table . CM capacitance manometer readings and Pirani Pirani gauge readings, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$T_{sh}$$\end{document} T sh shelf temperature, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$T_{air}$$\end{document} T air air temperature in the chamber. Measured product temperature: TC—thermocouple readings and color coded flexible sensor readings of 6R SCHOTT vials filled with 4ml 5% mannitol solution.

Article Snippet: The freeze-dryer is equipped with a vacuum capacitance manometer (MKS Instruments Baratron 622A) and a Pirani gauge pressure sensor (Granville-Phillips 275 Mini-Convectron).

Techniques: